US8343764B2 - Genes encoding glutamine synthetase and uses for plant improvement - Google Patents

Genes encoding glutamine synthetase and uses for plant improvement Download PDF

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US8343764B2
US8343764B2 US12/459,621 US45962109A US8343764B2 US 8343764 B2 US8343764 B2 US 8343764B2 US 45962109 A US45962109 A US 45962109A US 8343764 B2 US8343764 B2 US 8343764B2
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sense
protein
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plants
plant
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Mark Scott Abad
Marie A. Coffin
Barry S. Goldman
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Monsanto Technology LLC
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Monsanto Technology LLC
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis.
  • Folder 228pfamDir contains 288 Pfam Hidden Markov Models. Both folders were created on the disk on Jul. 2, 2009, having a total size of 24,465,168 bytes when measured in MS-WINDOWS® operating system.
  • transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds
  • Transgenic plants with improved traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property.
  • This invention provides plant cell nuclei with recombinant that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells.
  • Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17.
  • plant cells which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 426 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 850 and homologs thereof listed in Table 2.
  • Amino acid sequences of homologs are SEQ ID NO: 851 through 33634.
  • the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
  • transgenic plant cells and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants.
  • plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
  • the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein.
  • the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention.
  • the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
  • nuclei is cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.
  • the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
  • This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells.
  • the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of a such a protein
  • the method comprises (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is
  • the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound.
  • the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having one of the enhanced traits described above.
  • Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA
  • the method further comprises producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.
  • Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue.
  • Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
  • aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 426, 428, 429, 430, 524, 525, 541, 601, 602, 650, 651, 654, 655, 657, 660, 694, 698, 772, 801 or the corresponding Pfam identified in Table 16, i.e.
  • suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
  • Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency.
  • this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency.
  • methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop.
  • This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.
  • methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
  • the various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • the invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.
  • FIG. 1 is a consensus amino acid sequence of SEQ ID NO: 601 and homologs.
  • FIGS. 2 and 3 are plasmid maps.
  • SEQ ID NO:1-425 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;
  • SEQ ID NO:426-850 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-425;
  • SEQ ID NO:851-33634 are amino acid sequences of homologous proteins
  • SEQ ID NO:33635 is a consensus amino acid sequence.
  • SEQ ID NO:33637 is a DNA sequence of a plasmid base vector useful for soybean transformation.
  • the nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.
  • Transgenic seed means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein.
  • the term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.
  • Recombinant DNA a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit.
  • Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form.
  • a recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
  • Trait means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
  • a “control plant” is a plant without trait-improving recombinant DNA in its nucleus.
  • a control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA.
  • a suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein.
  • a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement.
  • a control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, the use of a limited number of control plants can cause a wide variation in the control dataset.
  • the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein.
  • Trait improvement includes, but is not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.
  • agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits.
  • Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
  • transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
  • Yield-limiting environment means the condition under which a plant would have the limitation on yield including environmental stress conditions.
  • Stress condition means a condition unfavorable for a plant, which adversely affect plant metabolism, growth and/or development.
  • a plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield.
  • water deficit stress used herein preferably refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants.
  • Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. Conditions which may result in water deficit stress include heat, drought, high salinity and PEG induced osmotic stress.
  • Cold stress means the exposure of a plant to a temperatures below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.
  • Nonrogen nutrient means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate.
  • ammonium as used herein means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc.
  • “Increased yield” of a transgenic plant of the present invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare.
  • maize yield can be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture.
  • a “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell.
  • Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium .
  • Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”.
  • a “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • an “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.
  • “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
  • operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.
  • Arabidopsis means plants of Arabidopsis thaliana.
  • Pfam refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.
  • profile HMMs Profile hidden Markov models
  • the present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells.
  • Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells.
  • Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides.
  • additional regulatory elements such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides.
  • Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
  • Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region.
  • UTR 3′ untranslated DNA region
  • useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens .
  • Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle.
  • a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle.
  • Table 1 provides a list of genes that provide recombinant DNA that was used in a model plant to discover associated improved traits and that can be used with homologs to define a consensus amino acid sequence for characterizing recombinant DNA for use in the nuclei of this invention. An understanding of Table 1 is facilitated by the following description of the headings:
  • NUC SEQ ID NO refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
  • PEP SEQ ID NO refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
  • Gene ID refers to an arbitrary name used to identify the particular DNA.
  • orientation refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.
  • DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:425, as well as the homologs of such DNA molecules.
  • a subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:425 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA.
  • Such variants may be naturally occurring, including DNA from homologous genes from the same or a different species, or may be non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques.
  • Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed.
  • a DNA useful in the present invention may have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).
  • the reference polynucleotide may be a full-length molecule or a portion of a longer molecule.
  • the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
  • Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 426 through SEQ ID NO: 850, as well as homologs of such proteins.
  • Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman.
  • a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared.
  • An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog.
  • a hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation.
  • homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity.
  • the relationship of homologs with amino acid sequences of SEQ ID NO: 851 to SEQ ID NO: 33634 to the proteins with amino acid sequences of SEQ ID NO: to 426 to SEQ ID NO: 850 is found in the listing of Table 2.
  • conserveed substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • a further aspect of the invention comprises proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
  • Protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.
  • promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens , caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters.
  • NOS nopaline synthase
  • OCS octopine synthase
  • caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters.
  • CaMV35S cauliflower mosaic virus
  • the promoters can include multiple “enhancer sequences” to assist in elevating gene expression.
  • enhancers are known in the art.
  • the expression of the selected protein may be enhanced.
  • These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence.
  • these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes.
  • the promoter element in the DNA construct be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide in water deficit conditions.
  • Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions.
  • Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize .
  • HSP17.5 heat shock protein 17.5 gene
  • HVA22 HVA22
  • Rab17 a cinnamic acid 4-hydroxylase
  • CA4H cinnamic acid 4-hydroxylase
  • promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al., (1996) Plant Mol Biol. 31(6): 1205-1216).
  • seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al.
  • Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
  • Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein.
  • Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression.
  • dsRNA double-stranded RNA
  • Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function.
  • transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained.
  • some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options.
  • Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
  • the present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with a multiple desired traits.
  • the combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
  • heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line.
  • it may be useful to target heterologous DNA insertion in order to achieve site-specific integration e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression.
  • site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
  • Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment.
  • Media refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism.
  • Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.
  • transgenic plants of this invention e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
  • Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes.
  • Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide.
  • Potentially transformed cells with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA in the nucleus.
  • Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference.
  • Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
  • Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in regeneration media and allowed to mature into plants.
  • Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO 2 , and 25-250 microeinsteins m ⁇ 2 s ⁇ 1 of light, prior to transfer to a greenhouse or growth chamber for maturation.
  • Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
  • Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide.
  • Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • “molecular biological” assays such as Southern and Northern blotting and PCR
  • biochemical assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function
  • Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
  • Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
  • a two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait.
  • Table 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
  • PEP SEQ ID which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.
  • construct_id is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
  • “annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
  • “description” refers to the description of the top BLAST hit.
  • e-value provides the expectation value for the BLAST hit.
  • identity refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
  • “traits” identify by two letter codes the confirmed improvement in a transgenic plant provided by the recombinant DNA.
  • the codes for improved traits are:
  • PEG which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen
  • SP which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.
  • subtilis str. 168 466 0 100 gi
  • Arabidopsis thaliana 467 0 100 gi
  • subtilis str. 168 713 0 87 gi
  • PCC 6803 721 0 100 gi
  • DS-Improvement of drought tolerance identified by a soil drought stress tolerance screen Drought or water deficit conditions impose mainly osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage.
  • the drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting.
  • the present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition.
  • Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3.
  • Such recombinant DNA may find particular use in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle.
  • transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition can also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
  • PEG-Improvement of drought tolerance identified by PEG induced osmotic stress tolerance screen Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130).
  • PEG polyethylene glycol
  • a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better drought conditions providing a higher yield potential as compared to control plants.
  • SS-Improvement of drought tolerance identified by high salinity stress tolerance screen Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought.
  • the present invention provides genes that help plants to maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3.
  • trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
  • HS-Improvement of drought tolerance identified by heat stress tolerance screen Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified by the present invention as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
  • CK and CS-Improvement of tolerance to cold stress Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential.
  • two screening conditions i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS)
  • CK cold shock tolerance screen
  • CS cold germination tolerance screen
  • the transgenic Arabidopsis plants were exposed to a constant temperature of 8° C. from planting until day 28 post plating.
  • the trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants.
  • transgenic plants were first grown under the normal growth temperature of 22° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen can survive better cold conditions providing a higher yield potential as compared to control plants.
  • PEP SEQ ID NO: 459 can be used to improve both salt stress tolerance and cold stress tolerance in plants.
  • plants transformed with PEP SEQ ID NO: 440 can resist heat stress, salt stress and cold stress.
  • the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
  • the present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 529 which can improve the plant early growth and development, and impart salt and cold tolerance to plants.
  • PEP SEQ ID NO: 529 which can improve the plant early growth and development, and impart salt and cold tolerance to plants.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
  • “Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity.
  • transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3 exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length.
  • the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant.
  • the seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
  • LL-Improvement of tolerance to shade stress identified in a low light screen The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased.
  • the present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants.
  • the transgenic plants generated by the present invention may be suitable for a higher density planting, thereby resulting increased yield per unit area.
  • the metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves.
  • All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
  • the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality.
  • the transgenic plants of the present invention may be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless.
  • the transgenic plants also may be advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
  • the present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits.
  • genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects.
  • Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides.
  • glyphosate herbicides glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides.
  • the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 425, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 426 through SEQ ID NO: 850.
  • the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 851 through SEQ ID NO: 33634.
  • the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.
  • the trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by the present invention are not limited to any particular plant species.
  • the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous.
  • they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications.
  • Of particular interest in the present invention are corn and soybean plants.
  • the recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention.
  • Other plants of interest in the present invention for production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart.
  • Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions.
  • Biomass measures may be made on greenhouse or field grown plants and may include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
  • Trait data on morphological changes may be collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil.
  • Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots.
  • Other enhanced traits may be identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance.
  • trait characteristics of harvested grain may be confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
  • hybrid yield in transgenic corn plants expressing genes of the present invention it may be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
  • Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein.
  • Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture.
  • Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
  • Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577).
  • the transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide.
  • This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
  • T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA).
  • Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 ⁇ mol/m 2 /s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
  • plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
  • seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
  • This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.
  • T2 seeds were plated on 1 ⁇ 2 ⁇ MS salts, 1/% phytagel, with 10 ⁇ g/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ⁇ 140 ⁇ mol/m 2 /s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
  • the seedling weight and root length were analyzed as quantitative responses according to example 1M.
  • the final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510).
  • the growth stage data was analyzed as a qualitative response according to example 1 L.
  • Table 5 provides a list of recombinant DNA constructs that improve heat tolerance in transgenic plants.
  • This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
  • T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m 2 . On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
  • the seedling weight and root length were analyzed as quantitative responses according to example 1M.
  • the final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510).
  • the growth stage data was analyzed as a qualitative response according to example 1 L.
  • Table 6 provides a list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants
  • T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, 1 ⁇ 2 ⁇ MS salts, 1% phytagel, and 10 ⁇ g/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
  • Seedling weight and root length were analyzed as quantitative responses according to example 1M.
  • the final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm.
  • the growth stage data was analyzed as a qualitative response according to example 1 L.
  • Table 7 provides a list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants.
  • This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
  • This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.
  • T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of 1 ⁇ 2 ⁇ Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% PhytagelTM (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH.
  • 1 ⁇ 2 ⁇ Gamborg's B/5 Basal Salt Mixture Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788
  • 1% PhytagelTM Sigma-Aldrich, P-8169
  • Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m 2 /s. At 28 days post plating, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
  • the root length at day 28 was analyzed as a quantitative response according to example 1M.
  • the growth stage at day 7 was analyzed as a qualitative response according to example 1 L.
  • Table 9 provides a list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants.
  • This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
  • T2 seeds were plated on glufosinate selection plates with 1 ⁇ 2 MS medium. Seeds were sown on 1 ⁇ 2 ⁇ MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m 2 /s, far/red ratio (655/665/725/735) ⁇ 0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1 L.
  • Table 10 provides a list of recombinant DNA constructs that improve shade tolerance in plants
  • the transgenic plants showed a trend of trait improvement as compared to the reference with p ⁇ 0.2.
  • “petiole length” if p ⁇ 0.05 and delta ⁇ 0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta ⁇ 0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth.
  • the transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
  • T2 seeds were plated on glufosinate selection plates and grown under standard conditions ( ⁇ 100 uE/m 2 /s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
  • Table 11 provides a list recombinant DNA constructs that improve early plant growth and development.
  • This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
  • Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft 3 . T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of ⁇ 5 per 21 ⁇ 2′′ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat.
  • Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ⁇ 200 ⁇ moles/m 2 /s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
  • the rosette radius was measured at day 25.
  • the silique length was measured at day 40.
  • the plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53.
  • the seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
  • Table 12 provides a list of recombinant DNA constructs that improve late plant growth and development.
  • Arabidopsis seedlings become chlorotic and have less biomass.
  • This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
  • T2 seeds were plated on glufosinate selection plates containing 0.5 ⁇ N-Free Hoagland's T 0.1 mM NH 4 NO 3 T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i. e., viable or non-viable) and root length. After 21 days of growth, plants were scored for BASTA resistance, visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
  • the seedling weight and root length were analyzed as quantitative responses according to example 1M.
  • the number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1 L.
  • the leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system.
  • a statistical logistic regression model was developed to predict an overall value based on five colors for each plant.
  • Table 13 provides a list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants.
  • the transgenic plants showed a trend of trait improvement as compared to the reference with p ⁇ 0.2.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Table 14 provides a list of responses that were analyzed as qualitative responses
  • a BLAST searchable “All Protein Database” is constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” is constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
  • NCBI National Center for Biotechnology Information
  • the Organism Protein Database is queried using amino acid sequences of SEQ ID NO: 426 through SEQ ID NO: 850 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value is compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism.
  • ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 426 and its homologs, through SEQ ID NO: 850 and its homologs.
  • Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty.
  • Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment.
  • the consensus sequence of SEQ ID NO: 601 and its 13 homologs was derived according to the procedure described above and is displayed in FIG. 1 .
  • This example illustrates the identification of amino acid domain by Pfam analysis.
  • the amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing.
  • the Pfam protein families for the proteins of SEQ ID NO: 425 through 850 are shown in Table 16.
  • the Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art.
  • Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 488 is characterized by two Pfam domains, i.e.
  • PABP PF00658.8 25 Poly-adenylate binding protein, unique domain p450 PF00067.11 ⁇ 105 Cytochrome P450 Aa_trans PF01490.7 ⁇ 128.4 Transmembrane amino acid transporter protein F-box PF00646.21 13.6 F-box domain LRR_2 PF07723.2 6 Leucine Rich Repeat LRR_2 PF07723.2 6 Leucine Rich Repeat FHA PF00498.14 25 FHA domain Bromodomain PF00439.14 8.9 Bromodomain PHD PF00628.17 25.9 PHD-finger SET PF00856.17 23.5 SET domain Abhydrolase_1 PF00561.10 10.3 alpha/beta hydrolase fold Epimerase PF01370.11 ⁇ 46.3 NAD dependent epimerase/dehydratase family 3Beta_HSD PF01073.8 ⁇ 135.9 3-beta hydroxysteroid dehydrogena
  • RRM_1 PF00076.11 20.7 RNA recognition motif (a.k.a. RRM, RBD, or RNP domain) RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) La PF05383.5 25 La domain RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) R
  • This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention.
  • Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions.
  • DNA of interest i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
  • Elements of an exemplary common expression vector, pMON82060 are illustrated in Table 18.
  • the exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art.
  • the DNA of interest are inserted in a expression vector at the insertion site between the intron 1 of rice act 1 gene and the termination sequence of PinII gene.
  • cassette I-Os.Act1 First intron and flanking UTR exon sequences from the rice actin 1 gene insertion site T-St.Pis4 The 3′ non-translated region of the 7084-8026 potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant P-CaMV.35S CaMV 35S promoter 8075-8398 selectable L-CaMV.35S 5′ UTR from the 35S RNA of CaMV marker CR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226 expression resistance to neomycin and kanamycin cassette T-AGRtu.nos A 3′ non-translated region of the 9255-9507 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA..
  • OR-Ec.oriV-RK2 The vegetative origin of replication from 567-963 in E. coli plasmid RK2.
  • OR-Ec.ori-ColE1 The minimal origin of replication from 3091-3679 the E. coli plasmid ColE1.
  • Tn7 adenylyltransferase 4210-4251 AAD(3′′)
  • CR-Ec.aadA- Coding region for Tn7 4252-5040 SPC/STR adenylyltransferase AAD(3′′)
  • conferring spectinomycin and streptomycin resistance AAD(3′′)
  • Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below.
  • This exemplary soybean transformation base vector illustrated in FIG. 3 was assembled using the technology known in the art. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.
  • T-AGRtu.nos A 3′ non-translated region 9466-9718 of the nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA.
  • Gene of P-CaMV.35S-enh Promoter for 35S RNA 1-613 interest from CaMV containing a expression duplication of the ⁇ 90 to ⁇ 350 cassette region.
  • insertion site T-Gb.E6-3b 3′ untranslated region 688-1002 from the fiber protein E6 gene of sea-island cotton; Agro B-AGRtu.right border Agro right border 1033-1389 transformation sequence, essential for transfer of T-DNA.
  • OR-Ec.oriV-RK2 The vegetative origin of 5661-6057 in E. coli replication from plasmid RK2.
  • OR-Ec.ori-ColE1 The minimal origin of 2945-3533 replication from the E. coli plasmid ColE1.
  • This example illustrates monocot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation of corn callus.
  • Corn plants of a readily transformable line are grown in the greenhouse and ears harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryos are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes.
  • Immature embryos are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformants are recovered 6 to 8 weeks after initiation of selection.
  • Plasmid vectors are prepared essentially as described in Example 5 for transforming into corn callus each of DNA identified in Table 1 and the corresponding DNA for the identified homologous genes identified in Table 2, by Agrobacterium -mediated transformation.
  • immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media, and paromomycin resistant callus identified as containing the recombinant DNA in an expression cassette.
  • Transgenic corn plants are regenerated from transgenic callus resulting from transformation on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber at 26 degrees C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity.
  • the plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g. for yield trials in the screens indicated above.
  • Populations of transgenic plants and seeds produced form transgenic plant cells from each transgenic event are screened as described in Example 7 below to identify the members of the population having the enhanced trait.
  • This example illustrates dicot plant transformation to produce nuclei of this transgenic in cells of transgenic plants by transformation of soybean tissue.
  • soybean seeds are germinated overnight and the meristem explants excised. The meristems and the explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed germination and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.
  • Trait positive shoots are harvested approximately 6-8 weeks post bombardment and placed into selective rooting media for 2-3 weeks.
  • Shoots producing roots are transferred to the greenhouse and potted in soil.
  • Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks.
  • Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil.
  • Populations of transgenic plants and seeds produced form transgenic plant cells from each transgenic event are screened as described in Example 7 below to identify the members of the population having the enhanced trait.
  • This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
  • transgenic seed and plants prepared in Examples 5 and 6 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait.
  • Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below.
  • Plant cell nuclei having recombinant DNA with each of the genes identified in Table 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.
  • Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area.
  • Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations.
  • Transgenic corn plants prepared in Example 5 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention.
  • Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention.
  • Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention. Selection for Increased Yield
  • transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.
  • Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control.
  • a useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant.
  • Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges.
  • Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field.
  • a pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events.
  • a useful planting density is about 30,000 plants/acre.
  • High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre.
  • Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 5 and 6 are screened for yield enhancement. At least one event from each of the corn and soybean plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.
  • the following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention.
  • This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle.
  • the primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points.
  • SIH shoot initial height
  • SWH shoot wilt height
  • SWM shoot wilted biomass
  • STM shoot turgid weight
  • SDM shoot dry biomass
  • Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for water use efficiency.
  • Transgenic plants having at least a 1% increase in GRG and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention.
  • Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance improvement (DS) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.
  • Cold germination assay Three sets of seeds are used for the assay.
  • the first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed.
  • the second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events.
  • the third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
  • Captan MAESTRO® 80DF Fungicide
  • Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9 ⁇ 8.9 cm) of a germination tray (54 ⁇ 36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C. for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty millilters of deionized water are added to each germination tray.
  • Convrion® growth chamber Convrion® growth chamber
  • Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
  • Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for water use efficiency.
  • Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention.
  • Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance improvement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.
  • the following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample.
  • Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan.
  • An NIR calibration for the analytes of interest is used to predict the values of an unknown sample.
  • the NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
  • Typical sample(s) Whole grain corn and soybean seeds
  • Analytical time to run method Less than 0.75 min per sample
  • Total elapsed time per run 1.5 minute per sample
  • Typical and minimum sample Corn typical: 50 cc; minimum 30 cc size: Soybean typical: 50 cc; minimum 5 cc
  • Typical analytical range Determined in part by the specific calibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%.
  • Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for increased protein and oil in seed.
  • Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.
  • genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQ ID NO: 426, 428, 429, 430, 524, 525, 541, 601, 602, 650, 651, 654, 655, 657, 660, 694, 698, 772, 801.

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Abstract

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. The present invention provides recombinant DNA molecules for expression of a protein, including a recombinant DNA molecule for expression of a glutamine synthetase.

Description

CROSS REFERENCE TO RATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/431,855 filed on May 10, 2006 now abandoned, which claims benefit under 35USC §119(e) of U.S. provisional application Ser. No. 60/679,917, filed May 10, 2005, and U.S. provisional application Ser. No. 60/723,596, filed Oct. 4, 2005; all of which are incorporated herein by reference in their entirety including the sequence listing, the extra table, and the computer program listing.
INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-R's, each containing the file named 38-21(53708)D_seqListing.txt, which is 97,936,808 bytes (measured in MS-WINDOWS) and was created on Jul. 2, 2009, are incorporated herein by reference in their entirety.
INCORPORATION OF TABLES
Two copies of Table 2 on CD-R's, each containing the file named 38-21(53708)D_table2.txt, which is 330, 968 bytes when measured in MS-WINDOWS® operating system, was created on Jul. 2, 2009, are incorporated herein by reference in their entirety.
TABLES
The patent contains table(s) that have been included at the end of the specification.
INCORPORATION OF COMPUTER PROGRAM LISTING
A Computer Program Listing with folders “hmmer-2.3.2” and “288pfamDir” is contained on a CD-R and is incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 228pfamDir contains 288 Pfam Hidden Markov Models. Both folders were created on the disk on Jul. 2, 2009, having a total size of 24,465,168 bytes when measured in MS-WINDOWS® operating system.
FIELD OF THE INVENTION
Disclosed herein are transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds
BACKGROUND OF THE INVENTION
Transgenic plants with improved traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property.
SUMMARY OF THE INVENTION
This invention provides plant cell nuclei with recombinant that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific embodiments of the invention plant cells are provided which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 426 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 850 and homologs thereof listed in Table 2. Amino acid sequences of homologs are SEQ ID NO: 851 through 33634. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In some aspects of the invention the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein. In yet another aspect of the invention the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
Yet other aspects of the invention provide nuclei is cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants. In other important embodiments for practice of various aspects of the invention in Argentina the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells. In some aspects of the invention the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of a such a protein More specifically the method comprises (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-425; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having one of the enhanced traits described above.
Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA The method further comprises producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.
Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
Aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 426, 428, 429, 430, 524, 525, 541, 601, 602, 650, 651, 654, 655, 657, 660, 694, 698, 772, 801 or the corresponding Pfam identified in Table 16, i.e. Histone, WD40, NPH3, FHA, PB1, ADH_zinc_N, NAPRTase, ADK_lid, p450, B56, DUF231, C2, DUF568, WD40, F-box, Pkinase, Terpene_synth, respectively. Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency. Alternatively methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
The various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
The invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a consensus amino acid sequence of SEQ ID NO: 601 and homologs.
FIGS. 2 and 3 are plasmid maps.
 DETAILED DESCRIPTION OF THE INVENTION
In the attached sequence listing:
SEQ ID NO:1-425 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;
SEQ ID NO:426-850 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-425;
SEQ ID NO:851-33634 are amino acid sequences of homologous proteins;
SEQ ID NO:33635 is a consensus amino acid sequence.
SEQ ID NO:33636 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and
SEQ ID NO:33637 is a DNA sequence of a plasmid base vector useful for soybean transformation.
The nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.
“Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression. In aspects of the invention where an improved trait is provided by expression of a protein, “gene” refers at least to coding nucleotide sequence for a protein or a function polypeptide fragment of a protein that imparts the trait. In aspects of the invention where an improved trait is provided by suppression of expression of an endogenous protein, “gene” refers to any part of the gene that can be a target for suppression.
“Transgenic seed” means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.
“Recombinant DNA” a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement. A control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used. As use herein a “reference” is a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, i.e., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait improvement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait improvement can be measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait improvement is only measured qualitatively. It is known that there can be a natural variation in a trait. Therefore, the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait improvement includes, but is not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.
Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means the condition under which a plant would have the limitation on yield including environmental stress conditions.
“Stress condition” means a condition unfavorable for a plant, which adversely affect plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” used herein preferably refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. Conditions which may result in water deficit stress include heat, drought, high salinity and PEG induced osmotic stress.
“Cold stress” means the exposure of a plant to a temperatures below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.
“Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. The term ammonium as used herein means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc.
“Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition. For example, a limiting nitrogen condition can refers to a growth condition with 50% or less of the conventional nitrogen inputs. “Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives optimal amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. One skilled in the art would recognize what constitute such soil, media and fertilizer inputs for most plant species.
“Shade stress” means a growth condition that has limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).
“Increased yield” of a transgenic plant of the present invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, maize yield can be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield can result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA can also be used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
“Expression” means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also refers to production of encoded protein from mRNA.
A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions. As used herein, “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
As used herein, “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
A “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.
Percent identity refers to the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
“Arabidopsis” means plants of Arabidopsis thaliana.
“Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 426 through SEQ ID NO: 850. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are Mito_carr, 6PGD, UBX, iPGM_N, WD40, Fer4, Enolase_C, DUF1639, PBP, PLAC8, Acyl-CoA_dh—1, Isoamylase_N, Acyl-CoA_dh—2, PC4, Sugar_tr, UCH, Enolase_N, HATPase_c, PRA-PH, Pkinase, SBP56, PEP-utilizers, SIS, PCI, DUF1644, Terpene_synth, Acyl-CoA_dh_M, Acyl-CoA_dh_N, Glutaminase, Lectin_legB, Dehydrin, MatE, Ank, 2-Hacid_dh_C, Chal_sti_synt_C, DUF1070, ATP-grasp—2, Arginase, HABP4_PAI-RBP1, ABC2_membrane, DUF1723, Glyco_hydro—1, MFS—1, ARD, PDT, HMA, Pro_isomerase, Ferric_reduct, PRA-CH, Aa_trans, ACT, LisH, PGM_PMM_II, Spermine_synth, zf-MYND, LRRNT—2, Ribul_P—3_epim, PGM_PMM_IV, NPH3, DapB_C, TPR—1, TPR—2, FAE1_CUT1_RppA, K_trans, F-box, Cyclin_C, ADK, NUDIX, NIR_SIR, PEPCK_ATP, La, DapB_N, MtN3_slv, FMO-like, TIM, FKBP_C, PMEI, Peptidase_C12, Cyclin_N, DUF568, Methyltransf—11, Methyltransf—12, DUF1677, DnaJ_C, BRAP2, IF2_N, Carboxyl_trans, mTERF, Glyoxalase, TMEM14, Mlo, Beta_elim_lyase, Pyr_redox_dim, Glyco_transf—8, Nicastrin, Flavodoxin—1, Epimerase, PTPA, Lipase—3, Pyr_redox—2, GSHPx, ELM2, PGI, Aminotran—1—2, ABC_tran, GRP, PGK, Oleosin, Sulfotransfer—1, EXS, DUF1325, AMP-binding, Arm, NTP_transferase, LSM, Metalloenzyme, Molybdop_Fe4S4, MFAP1_C, Aminotran—3, PHD, B56, DUF588, PSI_PsaF, zf-CCCH, HEAT, PALP, FH2, SapB—1, Ammonium_transp, MannoseP_isomer, NOP5NT, SapB—2, Pyr_redox, Pollen_allerg—1, Asp, DUF662, FHA, YjeF_N, COX5C, GTP_EFTU_D2, Ion_trans—2, PK, DUF231, FAD_binding—1, Hrf1, FAD_binding—4, FAD_binding—6, FAD_binding—8, CBS, Smr, aPHC, DUF241, Brix, Ras, Acetyltransf—1, NAF, SPX, Na_Ca_ex, C2, p450, PP2C, Histone, 2-Hacid_dh, SBF, CCT, BCNT, PK_C, Miro, CH, PfkB, ACP_syn_III_C, Sterol_desat, ADH_zinc_N, CS, Cys_Met_Meta_PP, Lactamase_B, Bromodomain, CDI, Linker_histone, DAO, Dicty_CAR, Aldo_ket_red, zf-AN1, Methyltransf—6, DUF1005, LEA—2, NIR_SIR_ferr, DUF260, Oxidored_FMN, DUF26, Lectin_C, Pec_lyase_C, Nop, TB2_DP1_HVA22, ADH_N, YGGT, NAPRTase, NAD_binding—1, DUF914, PGM_PMM_I, NAD_binding—2, AICARFT_IMPCHas, Auxin_inducible, NAD_binding—6, Anti-silence, RuBisCO_large, Response_reg, FeThRed_A, Di19, SNARE, PGM_PMM_III, Molydop_binding, efhand, zf-CCHC, GTP_EFTU, ARID, adh_short, Fibrillarin, RuBisCO_large_N, WWE, AA_permease, PABP, OMPdecase, RRM—1, U-box, OPT, TBC, MGS, DUF786, 3Beta_HSD, zf-UBP, zf-A20, DPBB—1, GDPD, PI-PLC-X, SEP, PI-PLC-Y, NOSIC, Glycolytic, SET, ADK_lid, Alpha-amylase, EB1, PGAM, Abhydrolase—1, Glyco_hydro—14, Lung—7-TM_R, Abhydrolase—3, TCTP, GATase—2, Gln-synt_C, 2OG-FeII_Oxy, Pribosyltran, MIF, CoA_trans, RCC1, Pkinase_Tyr, MIP, DnaJ, HSCB_C, Trehalose_PPase, LRR—1, Cupin—2, LRR—2, Glyco_hydro—28, Yip1, Trp_syntA, Sedlin_N, SGS, Aldedh, CK_II_beta, zf-C3HC4, GIDA, PB1, IMPDH, Carb_kinase, PurA, Molybdopterin, Nodulin-like, Tim17, Xan_ur'permease, Hist_deacetyl, RNA_pol_Rpb8, Agenet, Myb_DNA-binding, Glyoxal_oxid_N, Ribophorin_I, and FAE—3-kCoA_syn1.
Recombinant DNA Constructs
The present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
In a preferred embodiment, a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.
Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens. Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.
Table 1 provides a list of genes that provide recombinant DNA that was used in a model plant to discover associated improved traits and that can be used with homologs to define a consensus amino acid sequence for characterizing recombinant DNA for use in the nuclei of this invention. An understanding of Table 1 is facilitated by the following description of the headings:
“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
“construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.
“Gene ID” refers to an arbitrary name used to identify the particular DNA. “orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.
TABLE 1
NUC PEP
SEQ SEQ
ID ID Construct
NO NO Gene ID ID orientation
1 426 CGPG699 10919 ANTI-SENSE
2 427 CGPG567 11142 SENSE
3 428 CGPG267 11310 ANTI-SENSE
4 429 CGPG1179 11866 ANTI-SENSE
5 430 CGPG959 11937 ANTI-SENSE
6 431 CGPG2158 16218 SENSE
7 432 CGPG2446 17410 SENSE
8 433 CGPG1862 18401 SENSE
9 434 CGPG3014 18665 SENSE
10 435 CGPG1674 19141 SENSE
11 436 CGPG2680 19156 SENSE
12 437 CGPG3577 19621 SENSE
13 438 CGPG4065 19760 SENSE
14 439 CGPG3929 19857 SENSE
15 440 CGPG2488 70510 SENSE
16 441 CGPG3012 70605 SENSE
17 442 CGPG3162 70607 SENSE
18 443 CGPG607 70812 SENSE
19 444 CGPG4084 70933 SENSE
20 445 CGPG3917 70939 SENSE
21 446 CGPG4414 71319 SENSE
22 447 CGPG185 71446 SENSE
23 448 CGPG1679 71609 SENSE
24 449 CGPG271 71649 SENSE
25 450 CGPG4434 71814 SENSE
26 451 CGPG2198 71945 SENSE
27 452 CGPG5253 72005 SENSE
28 453 CGPG5231 72026 SENSE
29 454 CGPG4815 72533 SENSE
30 455 CGPG4859 72637 SENSE
31 456 CGPG1589 72782 SENSE
32 457 CGPG1826 72794 SENSE
33 458 CGPG3899 72919 SENSE
34 459 CGPG5610 72983 SENSE
35 460 CGPG5665 73025 SENSE
36 461 CGPG5697 73050 SENSE
37 462 CGPG5695 73092 SENSE
38 463 CGPG4862 73338 SENSE
39 464 CGPG4910 73344 SENSE
40 465 CGPG6541 73536 SENSE
41 466 CGPG1756 73644 SENSE
42 467 CGPG4927 73662 SENSE
43 468 CGPG5106 73672 SENSE
44 469 CGPG5167 73733 SENSE
45 470 CGPG1924 73846 SENSE
46 471 CGPG5201 73863 SENSE
47 472 CGPG1884 74059 SENSE
48 473 CGPG5089 74205 SENSE
49 474 CGPG5870 74321 SENSE
50 475 CGPG5888 74337 SENSE
51 476 CGPG1461 74388 SENSE
52 477 CGPG6743 74412 SENSE
53 478 CGPG6722 74445 SENSE
54 479 CGPG82 74522 SENSE
55 480 CGPG6761 74526 SENSE
56 481 CGPG6781 74576 SENSE
57 482 CGPG4914 74707 SENSE
58 483 CGPG5840 74743 SENSE
59 484 CGPG5980 74755 SENSE
60 485 CGPG1743 75202 SENSE
61 486 CGPG5434 75220 SENSE
62 487 CGPG5824 75226 SENSE
63 488 CGPG5879 75231 SENSE
64 489 CGPG5949 75239 SENSE
65 490 CGPG6096 75258 SENSE
66 491 CGPG6218 75270 SENSE
67 492 CGPG6226 75271 SENSE
68 493 CGPG7518 75355 SENSE
69 494 CGPG7654 75460 SENSE
70 495 CGPG6875 75847 SENSE
71 496 CGPG8259 75907 SENSE
72 497 CGPG8229 75927 SENSE
73 498 CGPG8224 75962 SENSE
74 499 CGPG1927 75984 SENSE
75 500 CGPG5962 76119 SENSE
76 501 CGPG5375 76209 SENSE
77 502 CGPG8949 76323 SENSE
78 503 CGPG8943 76346 SENSE
79 504 CGPG8896 76352 SENSE
80 505 CGPG8960 76360 SENSE
81 506 CGPG5891 76512 SENSE
82 507 CGPG7260 76575 SENSE
83 508 CGPG2647 76733 SENSE
84 509 CGPG6995 76741 SENSE
85 510 CGPG9046 76845 SENSE
86 511 CGPG9047 76857 SENSE
87 512 CGPG9083 76902 SENSE
88 513 CGPG9076 76913 SENSE
89 514 CGPG9108 76917 SENSE
90 515 CGPG9109 76929 SENSE
91 516 CGPG5933 77009 SENSE
92 517 CGPG6335 77022 SENSE
93 518 CGPG9174 77108 SENSE
94 519 CGPG9120 77125 SENSE
95 520 CGPG9200 77135 SENSE
96 521 CGPG9263 77219 SENSE
97 522 CGPG8087 77351 SENSE
98 523 CGPG385 14810 SENSE
99 524 CGPG1857 14835 ANTI-SENSE
100 525 CGPG1788 15419 ANTI-SENSE
101 526 CGPG1966 16223 SENSE
102 527 CGPG1908 17903 SENSE
103 528 CGPG3545 18279 SENSE
104 529 CGPG3557 18284 SENSE
105 530 CGPG3340 18332 SENSE
106 531 CGPG3431 18349 SENSE
107 532 CGPG3530 18623 SENSE
108 533 CGPG3119 19551 SENSE
109 534 CGPG3594 19627 SENSE
110 535 CGPG4031 19785 SENSE
111 536 CGPG4036 19944 SENSE
112 537 CGPG2384 70125 SENSE
113 538 CGPG1598 70504 SENSE
114 539 CGPG560 70830 SENSE
115 540 CGPG4002 70943 SENSE
116 541 CGPG1422 71711 ANTI-SENSE
117 542 CGPG4429 71962 SENSE
118 543 CGPG862 72352 SENSE
119 544 CGPG2251 72413 SENSE
120 545 CGPG646 72603 SENSE
121 546 CGPG2569 72676 SENSE
122 547 CGPG5507 72737 SENSE
123 548 CGPG5547 72742 SENSE
124 549 CGPG5517 72762 SENSE
125 550 CGPG5792 72993 SENSE
126 551 CGPG5766 73031 SENSE
127 552 CGPG5775 73091 SENSE
128 553 CGPG4809 73224 SENSE
129 554 CGPG4872 73340 SENSE
130 555 CGPG6420 73420 SENSE
131 556 CGPG6402 73489 SENSE
132 557 CGPG5073 73750 SENSE
133 558 CGPG5091 73751 SENSE
134 559 CGPG3570 73814 SENSE
135 560 CGPG4342 73855 SENSE
136 561 CGPG4483 73856 SENSE
137 562 CGPG1527 73929 SENSE
138 563 CGPG4351 74063 SENSE
139 564 CGPG4753 74072 SENSE
140 565 CGPG4757 74073 SENSE
141 566 CGPG4805 74075 SENSE
142 567 CGPG6624 74135 SENSE
143 568 CGPG3161 74313 SENSE
144 569 CGPG3770 74315 SENSE
145 570 CGPG6673 74427 SENSE
146 571 CGPG6702 74490 SENSE
147 572 CGPG21 74511 SENSE
148 573 CGPG6801 74531 SENSE
149 574 CGPG154 74532 SENSE
150 575 CGPG6762 74538 SENSE
151 576 CGPG6763 74550 SENSE
152 577 CGPG1467 74579 SENSE
153 578 CGPG6160 74644 SENSE
154 579 CGPG15 74703 SENSE
155 580 CGPG5825 74733 SENSE
156 581 CGPG5936 74749 SENSE
157 582 CGPG5974 74752 SENSE
158 583 CGPG1366 74773 SENSE
159 584 CGPG7390 74896 SENSE
160 585 CGPG7421 74976 SENSE
161 586 CGPG7446 74991 SENSE
162 587 CGPG6295 75289 SENSE
163 588 CGPG1476 76003 SENSE
164 589 CGPG1821 76074 SENSE
165 590 CGPG6975 76137 SENSE
166 591 CGPG6189 76220 SENSE
167 592 CGPG8868 76301 SENSE
168 593 CGPG8909 76318 SENSE
169 594 CGPG8951 76347 SENSE
170 595 CGPG5892 76513 SENSE
171 596 CGPG7171 76566 SENSE
172 597 CGPG6142 76719 SENSE
173 598 CGPG7212 76757 SENSE
174 599 CGPG9034 76891 SENSE
175 600 CGPG9270 77208 SENSE
176 601 CGPG1112 12116 ANTI-SENSE
177 602 CGPG1284 13053 ANTI-SENSE
178 603 CGPG1640 14733 SENSE
179 604 CGPG2136 16132 SENSE
180 605 CGPG3542 18276 SENSE
181 606 CGPG1691 70851 SENSE
182 607 CGPG4067 70941 SENSE
183 608 CGPG5335 72134 SENSE
184 609 CGPG27 72326 SENSE
185 610 CGPG3441 72978 SENSE
186 611 CGPG4375 73619 SENSE
187 612 CGPG5176 73737 SENSE
188 613 CGPG6637 74196 SENSE
189 614 CGPG661 74546 SENSE
190 615 CGPG869 74558 SENSE
191 616 CGPG6159 74643 SENSE
192 617 CGPG5931 75234 SENSE
193 618 CGPG6282 75278 SENSE
194 619 CGPG7671 75585 SENSE
195 620 CGPG1574 76063 SENSE
196 621 CGPG9294 77401 SENSE
197 622 CGPG2435 17808 SENSE
198 623 CGPG3336 18330 SENSE
199 624 CGPG3613 18409 SENSE
200 625 CGPG4168 19813 SENSE
201 626 CGPG6517 73580 SENSE
202 627 CGPG993 73935 SENSE
203 628 CGPG6631 74124 SENSE
204 629 CGPG5393 74725 SENSE
205 630 CGPG1313 76403 SENSE
206 631 CGPG8150 77366 SENSE
207 632 CGPG1661 72414 SENSE
208 633 CGPG6427 73409 SENSE
209 634 CGPG4906 73723 SENSE
210 635 CGPG5092 73747 SENSE
211 636 CGPG6146 74634 SENSE
212 637 CGPG6022 74769 SENSE
213 638 CGPG5883 75232 SENSE
214 639 CGPG7596 75429 SENSE
215 640 CGPG8248 75965 SENSE
216 641 CGPG8233 75975 SENSE
217 642 CGPG1573 76061 SENSE
218 643 CGPG7141 76155 SENSE
219 644 CGPG8941 76322 SENSE
220 645 CGPG6337 76440 SENSE
221 646 CGPG9005 76828 SENSE
222 647 CGPG9208 77136 SENSE
223 648 CGPG4348 77303 SENSE
224 649 CGPG8099 77352 SENSE
225 650 CGPG618 10913 ANTI-SENSE
226 651 CGPG251 10925 ANTI-SENSE
227 652 CGPG636 11357 SENSE
228 653 CGPG639 11358 SENSE
229 654 CGPG287 11432 ANTI-SENSE
230 655 CGPG893 11927 ANTI-SENSE
231 656 CGPG1122 12050 SENSE
232 657 CGPG657 12358 ANTI-SENSE
233 658 CGPG1489 13809 SENSE
234 659 CGPG2122 16877 SENSE
235 660 CGPG1683 18117 ANTI-SENSE
236 661 CGPG4685 71651 SENSE
237 662 CGPG4729 72449 SENSE
238 663 CGPG4880 72649 SENSE
239 664 CGPG5680 73107 SENSE
240 665 CGPG7317 74875 SENSE
241 666 CGPG4902 75079 SENSE
242 667 CGPG6720 75180 SENSE
243 668 CGPG5850 75228 SENSE
244 669 CGPG6010 75246 SENSE
245 670 CGPG7488 75375 SENSE
246 671 CGPG7592 75476 SENSE
247 672 CGPG7672 75502 SENSE
248 673 CGPG6923 75857 SENSE
249 674 CGPG6988 75873 SENSE
250 675 CGPG1357 75970 SENSE
251 676 CGPG5918 76115 SENSE
252 677 CGPG6020 76217 SENSE
253 678 CGPG7032 76287 SENSE
254 679 CGPG7069 76294 SENSE
255 680 CGPG8900 76305 SENSE
256 681 CGPG8923 76391 SENSE
257 682 CGPG6296 76438 SENSE
258 683 CGPG7053 76452 SENSE
259 684 CGPG7134 76563 SENSE
260 685 CGPG6951 77055 SENSE
261 686 CGPG6994 77059 SENSE
262 687 CGPG7019 77062 SENSE
263 688 CGPG7024 77063 SENSE
264 689 CGPG9255 77218 SENSE
265 690 CGPG9274 77256 SENSE
266 691 CGPG6130 77314 SENSE
267 692 CGPG8074 77344 SENSE
268 693 CGPG8081 77348 SENSE
269 694 CGPG172 10443 ANTI-SENSE
270 695 CGPG2482 17814 SENSE
271 696 CGPG3222 18537 SENSE
272 697 CGPG3259 18831 SENSE
273 698 CGPG1900 19044 ANTI-SENSE
274 699 CGPG4128 19950 SENSE
275 700 CGPG20 70221 SENSE
276 701 CGPG201 71110 SENSE
277 702 CGPG3420 71718 SENSE
278 703 CGPG4308 71838 SENSE
279 704 CGPG5244 72087 SENSE
280 705 CGPG5538 72729 SENSE
281 706 CGPG3738 73302 SENSE
282 707 CGPG6454 73484 SENSE
283 708 CGPG6530 73594 SENSE
284 709 CGPG5175 73736 SENSE
285 710 CGPG5756 74016 SENSE
286 711 CGPG5361 74266 SENSE
287 712 CGPG6709 74479 SENSE
288 713 CGPG6755 74549 SENSE
289 714 CGPG6765 74574 SENSE
290 715 CGPG6039 74607 SENSE
291 716 CGPG6158 74642 SENSE
292 717 CGPG3578 75015 SENSE
293 718 CGPG7528 75380 SENSE
294 719 CGPG7756 75560 SENSE
295 720 CGPG7663 75765 SENSE
296 721 CGPG8249 75977 SENSE
297 722 CGPG2232 76052 SENSE
298 723 CGPG5494 76109 SENSE
299 724 CGPG5947 76116 SENSE
300 725 CGPG7160 76158 SENSE
301 726 CGPG6262 76232 SENSE
302 727 CGPG6909 76269 SENSE
303 728 CGPG8884 76303 SENSE
304 729 CGPG8965 76474 SENSE
305 730 CGPG5861 76529 SENSE
306 731 CGPG7138 76625 SENSE
307 732 CGPG7246 76759 SENSE
308 733 CGPG5378 77026 SENSE
309 734 CGPG9168 77131 SENSE
310 735 CGPG1505 14944 SENSE
311 736 CGPG3614 18410 SENSE
312 737 CGPG3220 18536 SENSE
313 738 CGPG3062 19540 SENSE
314 739 CGPG3580 19624 SENSE
315 740 CGPG3725 70353 SENSE
316 741 CGPG592 71215 SENSE
317 742 CGPG4417 71320 SENSE
318 743 CGPG2276 73076 SENSE
319 744 CGPG4975 73666 SENSE
320 745 CGPG2790 73852 SENSE
321 746 CGPG4972 74080 SENSE
322 747 CGPG5140 74211 SENSE
323 748 CGPG19 74510 SENSE
324 749 CGPG6769 74527 SENSE
325 750 CGPG6770 74539 SENSE
326 751 CGPG737 74556 SENSE
327 752 CGPG6789 74577 SENSE
328 753 CGPG5374 74729 SENSE
329 754 CGPG5978 74753 SENSE
330 755 CGPG5979 74754 SENSE
331 756 CGPG7447 74908 SENSE
332 757 CGPG948 75107 SENSE
333 758 CGPG6052 75254 SENSE
334 759 CGPG7661 75741 SENSE
335 760 CGPG8261 75931 SENSE
336 761 CGPG5447 76207 SENSE
337 762 CGPG7068 76293 SENSE
338 763 CGPG5938 76517 SENSE
339 764 CGPG7100 76559 SENSE
340 765 CGPG9003 76804 SENSE
341 766 CGPG6299 77018 SENSE
342 767 CGPG9135 77115 SENSE
343 768 CGPG9144 77128 SENSE
344 769 CGPG9194 77158 SENSE
345 770 CGPG9202 77159 SENSE
346 771 CGPG9139 77163 SENSE
347 772 CGPG414 10923 ANTI-SENSE
348 773 CGPG1072 14836 SENSE
349 774 CGPG1986 15130 SENSE
350 775 CGPG2916 18214 SENSE
351 776 CGPG111 70236 SENSE
352 777 CGPG4317 70639 SENSE
353 778 CGPG4387 70668 SENSE
354 779 CGPG4492 70674 SENSE
355 780 CGPG597 70804 SENSE
356 781 CGPG345 70821 SENSE
357 782 CGPG4786 72505 SENSE
358 783 CGPG4998 72824 SENSE
359 784 CGPG5076 73283 SENSE
360 785 CGPG5104 73292 SENSE
361 786 CGPG6502 73578 SENSE
362 787 CGPG6630 74112 SENSE
363 788 CGPG5484 74249 SENSE
364 789 CGPG5394 74278 SENSE
365 790 CGPG6058 74359 SENSE
366 791 CGPG2090 74393 SENSE
367 792 CGPG6664 74414 SENSE
368 793 CGPG7706 75530 SENSE
369 794 CGPG7884 75752 SENSE
370 795 CGPG6965 75867 SENSE
371 796 CGPG1256 75910 SENSE
372 797 CGPG2297 76101 SENSE
373 798 CGPG6246 76431 SENSE
374 799 CGPG9037 76832 SENSE
375 800 CGPG1941 16023 SENSE
376 801 CGPG2055 16424 ANTI-SENSE
377 802 CGPG2427 17807 SENSE
378 803 CGPG386 70802 SENSE
379 804 CGPG393 70817 SENSE
380 805 CGPG609 70819 SENSE
381 806 CGPG4022 70908 SENSE
382 807 CGPG942 72351 SENSE
383 808 CGPG1800 73087 SENSE
384 809 CGPG4783 73223 SENSE
385 810 CGPG3257 73709 SENSE
386 811 CGPG1696 73804 SENSE
387 812 CGPG1982 73819 SENSE
388 813 CGPG3780 73851 SENSE
389 814 CGPG6602 74156 SENSE
390 815 CGPG6621 74194 SENSE
391 816 CGPG5493 74252 SENSE
392 817 CGPG5811 74317 SENSE
393 818 CGPG5902 74328 SENSE
394 819 CGPG6791 74506 SENSE
395 820 CGPG6778 74540 SENSE
396 821 CGPG6166 74650 SENSE
397 822 CGPG3735 74718 SENSE
398 823 CGPG5854 74745 SENSE
399 824 CGPG7451 74956 SENSE
400 825 CGPG5024 75076 SENSE
401 826 CGPG6054 75255 SENSE
402 827 CGPG6207 75269 SENSE
403 828 CGPG7620 75432 SENSE
404 829 CGPG7623 75468 SENSE
405 830 CGPG7775 75686 SENSE
406 831 CGPG73 75911 SENSE
407 832 CGPG2100 76064 SENSE
408 833 CGPG6026 76121 SENSE
409 834 CGPG7269 76193 SENSE
410 835 CGPG6361 76237 SENSE
411 836 CGPG6993 76281 SENSE
412 837 CGPG8899 76388 SENSE
413 838 CGPG6926 76557 SENSE
414 839 CGPG7172 76567 SENSE
415 840 CGPG7129 76624 SENSE
416 841 CGPG7276 76764 SENSE
417 842 CGPG9031 76855 SENSE
418 843 CGPG9105 76976 SENSE
419 844 CGPG9082 76985 SENSE
420 845 CGPG6212 77014 SENSE
421 846 CGPG9206 77112 SENSE
422 847 CGPG9151 77117 SENSE
423 848 CGPG9129 77138 SENSE
424 849 CGPG9224 77226 SENSE
425 850 CGPG9358 77418 SENSE

Recombinant DNA
DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:425, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:425 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA. Such variants may be naturally occurring, including DNA from homologous genes from the same or a different species, or may be non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a DNA useful in the present invention may have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
Homologs of the genes providing DNA demonstrated as useful in improving traits in model plants disclosed herein will generally have significant identity with the DNA disclosed herein. DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 80% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference polynucleotide may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 426 through SEQ ID NO: 850, as well as homologs of such proteins.
Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 851 to SEQ ID NO: 33634 to the proteins with amino acid sequences of SEQ ID NO: to 426 to SEQ ID NO: 850 is found in the listing of Table 2.
Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention comprises proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the trait-improving proteins disclosed provided herein will generally demonstrate significant sequence identity. Of particular interest are proteins having at least 50% sequence identity, more preferably at least about 70% sequence identity or higher, e.g., at least about 80% sequence identity with an amino acid sequence of SEQ ID NO: 426 through SEQ ID NO: 850. Of course useful proteins also include those with higher identity, e.g., 90% to 99% identity. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO: 426 through SEQ ID NO: 850.
Genes that are homologous to each other can be grouped into families and included in multiple sequence alignments. Then a consensus sequence for each group can be derived. This analysis enables the derivation of conserved and class-(family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. The consensus sequence can be used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship. Thus, the present invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.
Promoters
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.
Furthermore, the promoters can include multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. application Ser. No. 10/739,565, incorporated herein by reference.
In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al., (1996) Plant Mol Biol. 31(6): 1205-1216).
In some aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein. Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
Gene Stacking
The present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with a multiple desired traits. The combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
Transformation Methods
Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and may be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it may be useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
In practice DNA is introduced into only a small percentage of target cell nuclei in any one experiment. Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA in the nucleus. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m−2s−1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Discovery of Trait-Improving Recombinant DNA
To identify nuclei with recombinant DNA that confer improved traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table3 reports:
“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.
“construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
“annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
“description” refers to the description of the top BLAST hit.
“e-value” provides the expectation value for the BLAST hit.
“identity” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
“traits” identify by two letter codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:
“CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;
“CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;
“DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;
“PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
“HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;
“SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;
“LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;
“LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;
“PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;
“SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.
TABLE 3
PEP
SEQ annotation
ID e %
NO value identity GenBank id description trait
426 1.00E−35 100 gi|21553646| histone H2A CK
427 6.00E−59 100 gi|7327824| ref|NP_195785.1| CK
macrophage migration
inhibitory factor family
protein/MIF family protein
[Arabidopsis thaliana]
428 0 99 gi|28416663| ref|NP_180648.1| CK
transducin family protein/
WD-40 repeat family protein
429 2.00E−78 100 gi|23198326| gb|AAN15690.1|unknown CK
protein [Arabidopsis
thaliana]
430 0 100 gi|23297810| ref|NP_194910.2| CK LN
phototropic-responsive
NPH3 family protein
431 8.00E−48 100 gi|12248017| ref|NP_173542.1|small CK
nuclear ribonucleoprotein,
putative/snRNP, putative/
Sm protein, putative
432 1.00E−126 100 gi|16323514| ref|NP_187673.1| CK CS PP HS
diadenosine 5′,5′″-P1,P4-
tetraphosphate hydrolase,
putative [Arabidopsis
thaliana]
433 0 100 gi|30679613| ref|NP_172113.1|DNA- CK SS PEG
binding bromodomain-
containing protein Contains
similarity to a Ring3 protein
434 2.00E−48 100 gi|52354271| gb|AAC17827.1|similar to CK PP
late embryogenesis abundant
proteins
435 0 100 gi|20466169| ref|NP_564623.2| CK
sodium/calcium exchanger
family protein/calcium-
binding EF hand family
protein
436 2.00E−87 100 gi|19310829| ref|NP_179396.1|histone CK
H1-3 (HIS1-3)
437 1.00E−99 100 gi|21593872| ref|NP_563973.1| CK
lactoylglutathione lyase
family protein/glyoxalase I
family protein
438 1.00E−159 66 gi|15220367| ref|NP_176890.1|F-box CK CS HS
family protein [Arabidopsis
thaliana]
439 0 78 gi|9757954| ref|NP_199024.1|expressed CK
protein [Arabidopsis
thaliana]
440 5.00E−51 100 gi|21618078| ref|NP_176371.1| CK HS SS
postsynaptic protein-related
[Arabidopsis thaliana]
441 0 100 gi|24030234| ref|NP_027420.1|zinc CK CS SS LL PEG
finger (CCCH-type) family
protein [Arabidopsis
thaliana]
442 0 100 gi|21618116| ref|NP_567671.1|bile CK
acid:sodium symporter
family protein
443 1.00E−168 100 gi|16930405| ref|NP_850182.1|PUR CK PP HS SS
alpha-1 protein [Arabidopsis
thaliana]
444 0 83 gi|23397029| ref|NP_567709.1|26S CK CS PP
proteasome regulatory
subunit, putative (RPN7)
445 0 83 gi|30349498| ref|NP_850094.1|CBL- CK SS PEG
interacting protein kinase 3
(CIPK3)
446 0 100 gi|9294291| dbj|BAB02193.1|cytochrome CK PP
p450
447 0 100 gi|7270365| emb|CAB80133.1|cyclin CK PP HS SS PEG
delta-3
448 0 100 gi|31580811| gb|AAP51420.1|ferric- CK
chelate reductase
[Arabidopsis thaliana]
449 0 99 gi|8439909| ref|NP_563791.1|protein CK
phosphatase 2C family
protein/PP2C family
protein
450 0 100 gi|15238701| ref|NP_197894.1|cytochrome CK DS
P450 family protein
[Arabidopsis thaliana]
451 1.00E−178 100 gi|21553567| gb|AAM62660.1|unknown CK
[Arabidopsis thaliana]
452 0 100 gi|23198354| ref|NP_193140.1| CK HS
selenium-binding protein,
putative [Arabidopsis
thaliana]
453 0 100 gi|22655117| ref|NP_565411.2|calcium- CK CS HS SS LL
dependent protein kinase
isoform 6 (CPK6)
454 4.00E−83 100 gi|21537110| ref|NP_197849.1| CK LN
expressed protein
[Arabidopsis thaliana]
455 1.00E−122 100 gi|23397082| ref|NP_174418.1| CK PEG
photosystem I reaction
center subunit III family
protein
456 0 100 gi|30680112| ref|NP_179369.2|expressed CK
protein [Arabidopsis
thaliana]
457 0 100 gi|4678321| ref|NP_190390.1| CK
expressed protein
[Arabidopsis thaliana]
458 0 100 gi|3169180| ref|NP_179889.1|casein CK
kinase II alpha chain,
putative [Arabidopsis
thaliana]
459 0 100 gi|30725494| ref|NP_851182.1|protein CK SS
kinase family protein
[Arabidopsis thaliana]
460 0 100 gi|26986957| ref|NP_742382.1|4- CK DS
aminobutyrate
aminotransferase
[Pseudomonas putida
KT2440]
461 0 91 gi|37526249| ref|NP_929593.1|4- CK
aminobutyrate
aminotransferase (gamma-
amino-N-butyrate
transaminase) (GABA
transaminase)
462 0 83 gi|37527865| ref|NP_931210.1|glutamate CK
synthase [NADPH] small
chain (glutamate synthase
beta subunit) (NADPH-
GOGAT) (GLTS beta chain)
463 0 100 gi|23308229| ref|NP_175649.1|glycosyl CK PP
hydrolase family 1 protein/
beta-glucosidase, putative a
464 0 100 gi|30685484| ref|NP_849681.1|serine/threonine CK LL LN
protein phosphatase
2A (PP2A) 55 kDa
regulatory subunit B
[Arabidopsis thaliana]
465 0 99 gi|16080446| ref|NP_391273.1|phosphoglycerate CK PP
kinase [Bacillus
subtilis subsp. subtilis str.
168]
466 0 100 gi|6729016| gb|AAF27012.1|putative CK
SAR DNA-binding protein-1
[Arabidopsis thaliana]
467 0 100 gi|42569691| ref|NP_181253.2|transducin CK HS
family protein/WD-40
repeat family protein
468 5.00E−96 100 gi|38603990| ref|NP_177237.1|C2 CK
domain-containing protein
469 0 100 gi|22136622| ref|NP_193273.1| CK PP
cytochrome P450 family
protein [Arabidopsis
thaliana]
470 0 100 gi|22331742| ref|NP_190771.2|F-box CK
family protein/WD-40
repeat family protein
[Arabidopsis thaliana]
471 1.00E−162 100 gi|7021738| ref|NP_566517.1|expressed CK CS SS
protein [Arabidopsis
thaliana]
472 0 98 gi|10176804| dbj|BAB09992.1|serine/threonine CK SS LL
protein kinase-like
[Arabidopsis thaliana]
473 0 100 gi|28827534| ref|NP_568971.1|leucine- CK
rich repeat transmembrane
protein kinase, putative
474 0 100 gi|12321664| gb|AAG50866.1|protein CK
kinase, putative [Arabidopsis
thaliana]
475 1.00E−177 100 gi|2651299| ref|NP_181590.1|protein CK
kinase family protein
476 0 100 gi|56381947| ref|NP_564161.1| CK SS
phosphoglycerate/bisphosphoglycerate
mutase family
protein
477 0 100 gi|15075789| ref|NP_386871.1| CK
PROBABLE
PHOSPHOGLYCERATE
KINASE PROTEIN
478 0 96 gi|28872422| ref|NP_795041.1|glutamine CK CS
synthetase
479 0 100 gi|21595073| ref|NP_192046.1|mitogen- CK SS PEG
activated protein kinase,
putative/MAPK, putative
(MPK4)
480 0 100 gi|10174347| ref|NP_242595.1|glutamate CK
synthase (small subunit)
481 0 100 gi|16081086| ref|NP_391914.1|ornithine CK PP
aminotransferase
482 0 100 gi|18400003| ref|NP_564469.1|WD-40 CK HS SS LL PEG
repeat family protein
[Arabidopsis thaliana]
483 0 100 gi|4559336| ref|NP_181783.1|protein CK
kinase family protein
[Arabidopsis thaliana]
484 0 100 gi|11994319| ref|NP_188976.1|casein CK CS PP SS PEG
kinase, putative [Arabidopsis
thaliana]
485 0 100 gi|23507789| ref|NP_176390.1| CK LN
mitochondrial transcription
termination factor-related/
mTERF-related [Arabidopsis
thaliana]
486 1.00E−120 100 gi|10177463| dbj|BAB10854.1|unnamed CK LL
protein product [Arabidopsis
thaliana]
487 0 100 gi|15221219| ref|NP_177573.1|protein CK LL
kinase, putative [Arabidopsis
thaliana]
488 0 100 gi|15218854| ref|NP_174217.1|CBL- CK CS LL PEG
interacting protein kinase 18
(CIPK18)
489 0 100 gi|22136780| ref|NP_189420.1| CK PP LL
monodehydroascorbate
reductase, putative
[Arabidopsis thaliana]
490 1.00E−132 100 gi|17104619| gb|AAL34198.1|putative CK
glutathione peroxidase
[Arabidopsis thaliana]
491 0 100 gi|7268589| ref|NP_192114.1|sugar CK
transporter, putative
[Arabidopsis thaliana]
492 0 100 gi|56381949| ref|NP_200733.2|sugar CK
transporter family protein
[Arabidopsis thaliana]
493 7.00E−60 83 gi|31433365| ref|NP_922597.1|unknown CK SS LL
protein
494 1.00E−171 79 gi|34910712| ref|NP_916703.1|putative CK
nuclear RNA binding protein
A [Oryza sativa
495 1.00E−139 100 gi|51536564| ref|NP_189139.1|zinc finger CK PP
(C3HC4-type RING finger)
family protein
496 1.00E−136 98 gi|22995109| ref|ZP_00039591.1|COG0149: CK PEG
Triosephosphate
isomerase
497 0 74 gi|32488446| ref|XP_473139.1| CK
OSJNBa0004N05.3 [Oryza
sativa (japonica cultivar-
group)]
498 0 100 gi|23126040| ref|ZP_00107950.1|COG0155: CK
Sulfite reductase, beta
subunit (hemoprotein)
[Nostoc punctiforme PCC
73102]
499 0 100 gi|7287983| ref|NP_191594.1| CK PEG
armadillo/beta-catenin repeat
family protein/F-box
family protein [Arabidopsis
thaliana]
500 0 100 gi|9758604| dbj|BAB09237.1|beta- CK
amylase [Arabidopsis
thaliana]
501 0 100 gi|23463051| ref|NP_178065.1|inosine-5′- CK
monophosphate
dehydrogenase [Arabidopsis
thaliana]
502 1.00E−100 77 gi|55168250| gb|AAV44116.1|unknown CK
protein [Oryza sativa
(japonica cultivar-group)]
503 0 83 gi|50942175| ref|XP_480615.1|putative CK
aminoimidazolecarboximide
ribonucleotide
transformylase
504 1.00E−159 100 gi|10177927| ref|NP_199602.1| CK
respiratory burst oxidase
protein D (RbohD)/
NADPH oxidase
505 1.00E−172 86 gi|57899142| dbj|BAD87004.1|unknown CK
protein [Oryza sativa
(japonica cultivar-group)]
506 0 100 gi|15223469| ref|NP_171679.1|protein CK
kinase family protein
[Arabidopsis thaliana]
507 0 100 gi|3482918| ref|NP_172414.1|ATP- CK SS
citrate synthase (ATP-citrate
(pro-S-)-lyase/citrate
cleavage enzyme), putative
508 0 100 gi|23297150| ref|NP_567724.1|fibrillarin CK
2 (FIB2) [Arabidopsis
thaliana]
509 0 100 gi|7268631| ref|NP_193573.1|F-box CK LL PEG
family protein [Arabidopsis
thaliana]
510 6.00E−87 79 gi|21553981| ref|NP_564267.1|peptidyl- CK
prolyl cis-trans isomerase
cyclophilin-type family
protein [Arabidopsis
thaliana]
511 1.00E−151 68 gi|34913784| ref|NP_918239.1|pectate CK
lyase-like protein
512 4.00E−91 75 gi|53793523| dbj|BAD54684.1|unknown CK
protein [Oryza sativa
(japonica cultivar-group)]
513 1.00E−41 63 gi|50948309| ref|XP_483682.1|putative CK LL
auxin induced protein
514 1.00E−53 58 gi|56202166| dbj|BAD73644.1|60S CK CS PEG
ribosomal protein L18A-like
515 7.00E−85 93 gi|34910948| dbj|BAB84492.1|nuclear CK
movement protein-like
516 0 100 gi|6513938| ref|NP_566157.1|nodulin CK
family protein [Arabidopsis
thaliana]
517 1.00E−171 100 gi|6957722| ref|NP_186908.1|delta 7- CK LL
sterol-C5-desaturase,
putative [Arabidopsis
thaliana]
518 0 100 gi|17132051| ref|NP_486997.1| CK PP
hypothetical protein alr2957
[Nostoc sp. PCC 7120]
519 0 71 gi|34911116| ref|NP_916905.1|putative CK
glyoxal oxidase
520 6.00E−24 42 gi|4803925| ref|NP_565499.1|expressed CK SS
protein [Arabidopsis
thaliana]
521 0 81 gi|50934555| ref|XP_476805.1|unknown CK SS LN PEG
protein
522 8.00E−94 100 gi|26453028| ref|NP_197956.1| CK PP SS PEG
expressed protein
523 1.00E−167 100 gi|9795142| emb|CAB67657.2|splicing CS PP
factor-like protein
[Arabidopsis thaliana]
ref|NP_190918.3|zinc
knuckle (CCHC-type)
family protein [Arabidopsis
thaliana]
524 1.00E−146 100 gi|6642639| ref|NP_187382.1|forkhead- CS PEG
associated domain-
containing protein/FHA
domain-containing protein
525 0 100 gi|9279657| ref|NP_188451.1| CS PP HS LN
octicosapeptide/Phox/Bem1
p (PB1) domain-containing
protein [Arabidopsis
thaliana]
526 0 100 gi|9759010| dbj|BAB09537.1|unnamed CS
protein product [Arabidopsis
thaliana]
527 0 100 gi|18390671| ref|NP_563769.1|F-box CS PP HS SS
family protein
528 0 100 gi|12248033| ref|NP_172880.1| CS
phytochrome kinase,
putative [Arabidopsis
thaliana]
529 0 100 gi|21554404| ref|NP_179646.1|DNAJ CS PP SS
heat shock family protein
[Arabidopsis thaliana]
530 1.00E−111 100 gi|10177521| ref|NP_201441.1|dehydrin CS PEG
(RAB18) rab18 protein -
Arabidopsis thaliana
sp|P30185|DHR18_ARATH
Dehydrin Rab18
531 0 100 gi|20260458| dbj|BAB70612.1| CS SS
anthocyanin-related
membrane protein 1
532 0 100 gi|21553568| ref|NP_849480.1|expressed CS PP PEG
protein
533 0 100 gi|21553616| ref|NP_568089.1| CS
expressed protein
534 1.00E−158 100 gi|21554409| ref|NP_174469.1|histidine CS
biosynthesis bifunctional
protein (HISIE)
[Arabidopsis thaliana]
pir||T51812 phosphoribosyl-
AMP cyclohydrolase (EC
3.5.4.19)
535 1.00E−156 68 gi|50949063| ref|XP_493889.1|putative CS
protein kinase [Oryza sativa]
536 0 76 gi|31455393| emb|CAD92450.1|amino CS SS
acid permease 6 [Brassica
napus]
537 0 100 gi|6324421| sp|Q12068|GRE2_YEAST CS
NADPH-dependent
methylglyoxal reductase
GRE2 (Genes de respuesta a
estres protein 2)
538 0 99 gi|18401915| ref|NP_566612.1|histone CS
deacetylase family protein
[Arabidopsis thaliana]
539 0 100 gi|30685903| ref|NP_851041.1|zinc finger CS PEG
(CCCH-type) family protein
[Arabidopsis thaliana]
540 1.00E−134 68 gi|31432120| ref|NP_921503.1|putative CS PP
polyprotein [Oryza sativa
(japonica cultivar-group)]
541 0 100 gi|22136556| emb|CAB80464.1| CS
cinnamyl-alcohol
dehydrogenase ELI3-2
[Arabidopsis thaliana]
542 0 100 gi|7413528| ref|NP_196086.1| CS
cytochrome P450, putative
[Arabidopsis thaliana]
543 0 99 gi|42568440| ref|NP_199845.2|DNA CS PEG
repair protein-related
[Arabidopsis thaliana]
544 0 100 gi|30102478| ref|NP_195917.1|hydrolase, CS PEG
alpha/beta fold family
protein [Arabidopsis
thaliana]
545 0 100 gi|21280881| ref|NP_176968.1|glycerate CS HS SS PEG
dehydrogenase/NADH-
dependent hydroxypyruvate
reductase
546 0 100 gi|10177967| ref|NP_198904.1|WD-40 CS PEG
repeat family protein/zfwd3
protein (ZFWD3)
[Arabidopsis thaliana]
547 0 100 gi|7269306| ref|NP_567705.1|ubiquitin- CS
specific protease 16, putative
(UBP16)
548 0 99 gi|6324066| sp|P53845|YN03_YEAST CS
Hypothetical 35.5 kDa
protein in PIK1-POL2
intergenic region
549 0 99 gi|6323074| ref|NP_013146.1|Microtubule- CS
associated protein (MAP)
of the XMAP215/Dis1
family; regulates
microtubule dynamics
during spindle orientation
and metaphase chromosome
alignment; interacts with
spindle pole body
component Spc72p
[Saccharomyces cerevisiae]
550 0 100 gi|6324990| ref|NP_015058.1|Dicarboxylic CS PP
amino acid permease,
mediates high-affinity and
high-capacity transport of L-
glutamate and L-aspartate;
also a transporter for Gln,
Asn, Ser, Ala, and Gly
[Saccharomyces cerevisiae]
551 0 100 gi|51013603| sp|P50947|YNJ7_YEAST CS PEG
Hypothetical 37.0 kDa
protein in RAS2-RPS7B
intergenic region
552 0 100 gi|6319413| ref|NP_009495.1|Shp1p CS
[Saccharomyces cerevisiae]
emb|CAA80789.1|
YBLO515 [Saccharomyces
cerevisiae]
emb|CAA84878.1|SHP1
553 0 100 gi|22136824| ref|NP_197455.1| CS PP
expressed protein
[Arabidopsis thaliana]
554 0 100 gi|22655036| ref|NP_566453.1|WD-40 CS
repeat family protein
[Arabidopsis thaliana]
555 0 100 gi|48771699| ref|ZP_00276041.1|COG1062: CS
Zn-dependent alcohol
dehydrogenases, class III
[Ralstonia metallidurans
CH34]
556 0 100 gi|49176169| ref|NP_416429.3|D-cysteine CS
desulfhydrase, PLP-
dependent enzyme cysteine
desulfhydrase, PLP-
dependent enzyme
557 0 100 gi|9294512| gb|AAK21273.1|aberrant CS PP SS
lateral root formation 5
[Arabidopsis thaliana]
ref|NP_566730.1|MATE
efflux family protein
[Arabidopsis thaliana]
558 0 100 gi|31711972| ref|NP_175641.1|protein CS SS
kinase family protein/C-
type lectin domain-
containing protein
559 8.00E−61 100 gi|21592962| ref|NP_564579.1|expressed CS SS
protein [Arabidopsis
thaliana]
560 0 100 gi|30102452| ref|NP_195770.1| CS LL
mitochondrial substrate
carrier family protein
[Arabidopsis thaliana]
561 0 100 gi|6056406| gb|AAF02870.1|Hypothetical CS
protein [Arabidopsis
thaliana]
562 1.00E−143 100 gi|21689635| emb|CAB78986.1|lectin like CS SS
protein [Arabidopsis
thaliana]
563 0 100 gi|22135769| ref|NP_188127.1| CS
oxidoreductase, zinc-binding
dehydrogenase family
protein
564 6.00E−91 100 gi|21553433| ref|NP_199669.1|FK506- CS PEG
binding protein 2-2
(FKBP15-2)/immunophilin/
peptidyl-prolyl cis-trans
isomerase/rotamase
565 1.00E−151 100 gi|21592742| ref|NP_195534.1|expansin, CS PP
putative (EXP20)
566 8.00E−64 100 gi|21537125| ref|NP_196975.1|expressed CS
protein [Arabidopsis
thaliana]
567 1.00E−49 100 gi|16331623| ref|NP_442351.1|hypothetical CS LN
protein ssl0353
[Synechocystis sp. PCC
6803]
568 0 100 gi|21618054| ref|NP_564383.1|ABC CS
transporter family protein
[Arabidopsis thaliana]
569 0 100 gi|7321037| ref|NP_192879.1| CS PP SS
ARID/BRIGHT DNA-
binding domain-containing
protein/ELM2 domain-
containing protein/Myb-
like DNA-binding domain-
containing protein
570 1.00E−44 100 gi|58299| emb|CAA48415.1|unnamed CS
protein product [synthetic
construct]
571 0 99 gi|10172815| dbj|BAB03922.1|glycine CS PEG
betaine aldehyde
dehydrogenase [Bacillus
halodurans C-125]
572 1.00E−165 100 gi|3928103| ref|NP_181434.1|aquaporin, CS PP
putative [Arabidopsis
thaliana]
573 0 91 gi|37524126| ref|NP_927470.1|phosphoenolpyruvate CS
carboxykinase
[ATP] [Photorhabdus
luminescens subsp.
laumondii TTO1]
574 0 100 gi|9293860| ref|NP_564217.1|lysine and CS
histidine specific transporter,
putative [Arabidopsis
thaliana]
575 0 100 gi|10175358| ref|NP_243603.1|1- CS
pyrroline-5-carboxylate
dehydrogenase [Bacillus
halodurans C-125]
576 0 100 gi|10175785| ref|NP_244029.1|pyruvate CS PP
kinase [Bacillus halodurans
C-125]
577 8.00E−45 100 gi|9758890| dbj|BAB09466.1|auxin- CS
induced protein-like
[Arabidopsis thaliana]
578 1.00E−108 100 gi|17104761| ref|NP_193699.1|Ras- CS LN
related GTP-binding protein,
putative [Arabidopsis
thaliana]
579 0 100 gi|5915859| sp|O22203|C98A3_ARATH CS
Cytochrome P450 98A3
580 0 100 gi|6729015| ref|NP_187156.1|protein CS SS PEG
kinase family protein
[Arabidopsis thaliana]
581 0 100 gi|21689671| ref|NP_195397.1| CS PP SS
transporter-related
[Arabidopsis thaliana]
582 0 100 gi|20148451| ref|NP_564797.1|flavin- CS SS PEG
containing monooxygenase
family protein/FMO family
protein
583 0 100 gi|21280977| gb|AAM45011.1|putative CS
protein kinase [Arabidopsis
thaliana]
584 1.00E−116 82 gi|27452901| gb|AAO15285.1|Putative CS
dihydrodipicolinate
reductase-like protein
[Oryza sativa (japonica
cultivar-group)]
585 1.00E−123 99 gi|23113896| ref|ZP_00099233.1|COG0036: CS
Pentose-5-phosphate-3-
epimerase
[Desulfitobacterium
hafniense DCB-2]
586 8.00E−95 99 gi|16330731| |YCF3_SYNY3 CS
Photosystem I assembly
protein ycf3
587 0 100 gi|21700831| ref|NP_191332.2|protein CS DS SS PEG
kinase, putative [Arabidopsis
thaliana]
588 0 100 gi|4415911| gb|AAD20142.1|putative CS
poly(A) binding protein
[Arabidopsis thaliana]
589 0 100 gi|5733869| gb|AAD49757.1|Contains F- CS
box domain PF|00646.
[Arabidopsis thaliana]
590 1.00E−149 100 gi|29824243| ref|NP_192170.1|tryptophan CS
synthase, alpha subunit,
putative [Arabidopsis
thaliana]
591 0 100 gi|26449849| ref|NP_198924.1| CS HS
glycerophosphoryl diester
phosphodiesterase family
protein
592 0 78 gi|56783874| dbj|BAD81286.1|putative CS
dual specificity kinase 1
593 0 100 gi|6321857| ref|NP_011933.1|Ssf1p CS DS
[Saccharomyces cerevisiae]
sp|P38789|SSF1_YEAST
Ribosome biogenesis protein
SSF1
594 1.00E−180 89 gi|50906987| ref|XP_464982.1|lipase class CS
3-like [Oryza sativa
(japonica cultivar-group)]
dbj|BAD21500.1|lipase
class 3-like [Oryza sativa
(japonica cultivar-group)]
595 0 100 gi|2558659| gb|AAB81672.1|putative CS HS PEG
protein kinase [Arabidopsis
thaliana]
596 0 100 gi|9758547| ref|NP_201503.1| CS
expressed protein
[Arabidopsis thaliana]
597 0 100 gi|4678335| ref|NP_190376.1|L- CS SS
galactono-1,4-lactone
dehydrogenase, putative
598 1.00E−173 100 gi|21553608| ref|NP_564800.1|expressed CS
protein [Arabidopsis
thaliana]
599 0 79 gi|50912531| ref|XP_467673.1|putative CS DS SS PEG
nicastrin [Oryza sativa
(japonica cultivar-group)]
600 0 80 gi|50898642| ref|XP_450109.1|nodulation CS PP SS
receptor kinase-like protein
601 5.00E−74 100 gi|30693828| ref|NP_850693.1|expressed DS
protein [Arabidopsis
thaliana]
602 0 100 gi|25054896| ref|NP_179923.2| DS
nicotinate
phosphoribosyltransferase
family protein/NAPRTase
family protein [Arabidopsis
thaliana]
603 1.00E−121 100 gi|6006854| gb|AAF00630.1|hypothetical DS LN
protein [Arabidopsis
thaliana]
604 0 100 gi|6862917| ref|NP_566273.1|heavy- DS
metal-associated domain-
containing protein
605 0 100 gi|56382003| ref|NP_177794.1|12- DS
oxophytodienoate reductase
(OPR1) [Arabidopsis
thaliana]
606 0 100 gi|21280825| gb|AAM45040.1|putative DS
AtMlo-h1 protein
[Arabidopsis thaliana]
607 0 61 gi|22136432| ref|NP_191438.2|glycosyl DS SS
transferase family 8 protein
[Arabidopsis thaliana]
608 0 69 gi|22136270| ref|NP_190235.1| DS
armadillo/beta-catenin repeat
family protein/U-box
domain-containing family
protein
609 0 100 gi|4678377| ref|NP_193087.1| DS
ammonium transporter 1,
member 1 (AMT1.1)
610 1.00E−143 99 gi|27754217| ref|NP_187413.1|expressed DS LN PEG
protein [Arabidopsis
thaliana]
611 1.00E−123 100 gi|21554344| ref|NP_198627.1|ASF1- DS PP HS
like anti-silencing family
protein [Arabidopsis
thaliana]
612 0 77 gi|25402857| pir||A86318protein DS PP
F15H18.11 [imported] -
Arabidopsis thaliana
gb|AAF25996.1|F15H18.11
613 1.00E−139 99 gi|16329950| ref|NP_440678.1|hypothetical DS
protein slr1900
[Synechocystis sp. PCC
6803]
614 0 100 gi|30725526| gb|AAP37785.1|At4g24520 DS SS
[Arabidopsis thaliana]
emb|CAB79362.1|NADPH-
ferrihemoprotein reductase
ATR1
615 0 98 gi|3335345| gb|AAC27147.1|Contains DS LL PEG
similarity to ABC
transporter gb|1651790 from
Synechocystis sp.
616 1.00E−120 100 gi|38603932| ref|NP_193578.1|Ras- DS
related GTP-binding protein,
putative [Arabidopsis
thaliana]
617 0 100 gi|15810337| ref|NP_565310.1|expressed DS PEG
protein [Arabidopsis
thaliana]
618 0 100 gi|8978074| ref|NP_199518.1|protein DS
kinase, putative [Arabidopsis
thaliana]
619 0 75 gi|9294678| dbj|BAB03027.1|glutamine- DS
fructose-6-phosphate
transaminase 2 [Arabidopsis
thaliana]
620 0 100 gi|42567142| ref|NP_194265.2|EXS DS
family protein/
ERD1/XPR1/SYG1 family
protein [Arabidopsis
thaliana] gb|AAR99486.1|
PHO1-like protein
[Arabidopsis thaliana]
621 1.00E−151 89 gi|50938765| ref|XP_478910.1|serine/threonine DS SS PEG
protein kinase PKPA-
like protein [Oryza sativa
622 0 100 gi|16323410| gb|AAD49980.1|Similar to HS
gb|AF110333 PrMC3
protein from Pinus radiata
and is a member of
PF|00135 Carboxylesterases
family.
623 7.00E−86 100 gi|23397128| ref|NP_178620.1|glycine- HS SS
rich protein (GRP)
[Arabidopsis thaliana]
624 1.00E−104 100 gi|24417354| ref|NP_564833.1| HS
expressed protein
[Arabidopsis thaliana]
625 1.00E−48 57 gi|21553397| ref|NP_180326.1|zinc HS
finger (AN1-like) family
protein [Arabidopsis
thaliana]
626 0 100 gi|16130575| ref|NP_417147.1|succinate- HS
semialdehyde
dehydrogenase I, NADP-
dependent [Escherichia coli
K12]
627 0 100 gi|15218645| ref|NP_176713.1|cytochrome HS PEG
P450, putative
[Arabidopsis thaliana]
628 0 100 gi|16329269| ref|NP_439997.1|hypothetical HS
protein slr0731
[Synechocystis sp. PCC
6803]
629 0 99 gi|12322845| gb|AAG51407.1|putative HS
cysteine synthase; 39489-37437
[Arabidopsis thaliana]
630 1.00E−126 100 gi|28827686| gb|AAO50687.1|unknown HS PEG
protein [Arabidopsis
thaliana]
631 1.00E−143 100 gi|23197714| ref|NP_196259.21|DNAJ HS SS LL
heat shock N-terminal
domain-containing protein
632 0 100 gi|21554091| gb|AAM63172.1|putative LL
integral membrane protein
[Arabidopsis thaliana]
633 0 100 gi|16329756| ref|NP_440484.1|formaldehyde LL
dehydrogenase
(glutathione)
634 0 100 gi|26450489| ref|NP_190148.1| LL
transducin family protein/
WD-40 repeat family protein
635 0 100 gi|3980410| ref|NP_180485.1|lectin LL LN
protein kinase, putative
[Arabidopsis thaliana]
636 1.00E−153 100 gi|6572061| ref|NP_190708.1| LL LN
expressed protein
[Arabidopsis thaliana]
637 1.00E−141 100 gi|22328141| ref|NP_201402.2|heterogeneous LL LN
nuclear
ribonucleoprotein, putative/
hnRNP, putative
638 0 100 gi|15219657| ref|NP_176819.1|protein LL
kinase family protein
[Arabidopsis thaliana]
639 5.00E−53 55 gi|34906632| ref|NP_914663.1|P0431G06.11 LL
[Oryza sativa (japonica
cultivar-group)]
640 1.00E−169 100 gi|21674686| ref|NP_662751.1|6- LL
phosphogluconate
dehydrogenase,
decarboxylating, putative
[Chlorobium tepidum TLS]
641 1.00E−69 96 gi|32490260| ref|XP_473078.1| LL PEG
OSJNBa0014K14.9 [Oryza
sativa (japonica cultivar-
group)]
642 0 100 gi|52354393| gb|AAU44517.1|hypothetical LL PEG
protein AT4G22980
[Arabidopsis thaliana]
643 2.00E−90 100 gi|9758347| ref|NP_200586.1|expressed LL LN
protein [Arabidopsis
thaliana]
644 0 82 gi|50946759| ref|XP_482907.1|putative LL LN
glycoprotein 3-alpha-L-
fucosyltransferase
645 1.00E−131 83 gi|7269121| emb|CAB79230.1|predicted LL
protein [Arabidopsis
thaliana]
646 1.00E−130 88 gi|50929089| ref|XP_474072.1|OSJNBb0079B02.14 LL LN
[Oryza sativa
(japonica cultivar-group)]
647 0 100 gi|10176523| ref|NP_244766.1|acetyl- LL
CoA: acetoacetyl-CoA
transferase [Bacillus
halodurans C-125]
648 0 100 gi|34365735| ref|NP_566384.1| LL
xanthine/uracil permease
family protein [Arabidopsis
thaliana]
649 1.00E−84 100 gi|10177539| ref|NP_201459.1|expressed LL
protein [Arabidopsis
thaliana]
650 1.00E−137 100 gi|21537398| ref|NP_201145.1|adenylate LN
kinase [Arabidopsis
thaliana]
651 0 100 gi|10177005| ref|NP_851136.1| LN
cytochrome P450 family
protein [Arabidopsis
thaliana]
652 0 99 gi|9758597| ref|NP_851196.1|outward LN
rectifying potassium channel
(KCO1) [Arabidopsis
thaliana]
653 1.00E−126 100 gi|20453405| ref|NP_180901.1|plastid LN
developmental protein DAG,
putative [Arabidopsis
thaliana]
654 0 100 gi|7378631| ref|NP_195967.1| LN
serine/threonine protein
phosphatase 2A (PP2A)
regulatory subunit B′
(B′alpha)
655 0 100 gi|50058955| gb|AAT69222.1|hypothetical LN
protein At2g30900
[Arabidopsis thaliana]
656 3.00E−36 100 gi|26453058| ] ref|NP_191804.1| LN
expressed protein
[Arabidopsis thaliana]
657 0 100 gi|28973131| ref|NP_190313.1| LN
phosphoinositide-specific
phospholipase C family
protein
658 0 100 gi|23297411| gb|AAN12963.1|enolase (2- LN
phospho-D-glycerate
hydroylase) [Arabidopsis
thaliana]
659 1.00E−157 100 gi|22136938| ref|NP_566528.1|expressed LN
protein [Arabidopsis
thaliana]
660 0 100 gi|9294186| ref|NP_566763.1|auxin- LN
responsive family protein
[Arabidopsis thaliana]
661 0 100 gi|7269372| ref|NP_194252.1| LN
transporter, putative
[Arabidopsis thaliana]
662 3.00E−93 100 gi|28393887| ref|NP_182046.1|auxin- LN
responsive protein-related
[Arabidopsis thaliana]
663 0 100 gi|4725942| ref|NP_192980.1|trehalose- LN
6-phosphate phosphatase,
putative
664 0 100 gi|15074657| ref|NP_385829.1| LN
PROBABLE
AMINOTRANSFERASE
PROTEIN
665 1.00E−179 81 gi|35215000| ref|NP_927371.1| LN
glutathione dependent
formaldehyde
dehydrogenase
666 1.00E−142 100 gi|23507799| ref|NP_565973.1|LOB LN
domain protein 16/lateral
organ boundaries domain
protein 16
667 0 99 gi|48731145| ref|ZP_00264891.1|COG1012: LN
NAD-dependent aldehyde
dehydrogenases
[Pseudomonas fluorescens
PfO-1]
668 0 99 gi|15223033| ref|NP_177763.1|protein LN
kinase, putative [Arabidopsis
thaliana]
669 0 100 gi|22137136| ref|NP_181639.1|RNA LN
recognition motif (RRM)-
containing protein
670 0 100 gi|50904773| ref|XP_463875.1|putative LN
iron-phytosiderophore
transporter protein yellow
stripe 1
671 5.00E−40 48 gi|15232064| ref|NP_189339.1|expressed LN
protein [Arabidopsis
thaliana]
672 0 88 gi|51535369| dbj|BAD37240.1|putative LN
phosphotyrosyl phosphatase
activator [Oryza sativa
(japonica cultivar-group)]
673 8.00E−93 100 gi|21555349| ref|NP_190798.1|ATP LN
synthase D chain-related
[Arabidopsis thaliana]
674 0 100 gi|9795597| ref|NP_173294.1| LN
sulfotransferase family
protein [Arabidopsis
thaliana]
675 0 100 gi|42567054| ref|NP_194058.2|protein LN
kinase family protein
[Arabidopsis thaliana]
676 0 100 gi|31711846| ref|NP_177412.1| LN
cinnamyl-alcohol
dehydrogenase, putative
677 0 100 gi|23308375| ref|NP_851141.1|RNA LN
recognition motif (RRM)-
containing protein
678 0 100 gi|7329658| emb|CAB82755.1|protein LN
kinase ATN1-like protein
[Arabidopsis thaliana]
679 0 100 gi|22655386| ref|NP_197288.1|cation LN
exchanger, putative (CAX7)
[Arabidopsis thaliana]
680 1.00E−162 100 gi|20197230| gb|AAM14984.1|high LN
affinity K+ transporter
(AtKUP1 AtKT1p)
[Arabidopsis thaliana]
681 0 100 gi|15219169| ref|NP_175713.1|protein LN
kinase family protein
[Arabidopsis thaliana]
682 0 100 gi|9758951| ref|NP_568809.2|protein LN
kinase family protein
[Arabidopsis thaliana]
683 0 100 gi|7573368| ref|NP_196762.1|expressed LN
protein [Arabidopsis
thaliana]
684 0 100 gi|20259017| ref|NP_200323.1| LN
expressed protein
[Arabidopsis thaliana]
685 6.00E−80 100 gi|30793811| ref|NP_191519.1|DNA- LN PEG
directed RNA polymerase I,
II, and III, putative
686 0 100 gi|7268609| ref|NP_193550.1|outward LN
rectifying potassium
channel, putative (KCO6)
687 1.00E−111 71 gi|7270315| ref|NP_195093.1|L- LN
galactose dehydrogenase (L-
GalDH) [Arabidopsis
thaliana]
688 5.00E−98 100 gi|29824277| ref|NP_568016.1|expressed LN
protein [Arabidopsis
thaliana]
689 1.00E−170 69 gi|7269325| emb|CAB79384.1|protein LN
kinase (AFC2) [Arabidopsis
thaliana]
690 3.00E−96 79 gi|57899263| dbj|BAD87508.1|putative LN
calcyclin-binding protein
[Oryza sativa (japonica
cultivar-group)]
691 1.00E−164 100 gi|11994637| emb|CAD44270.1| LN
monomeric G-protein
[Arabidopsis thaliana]
692 1.00E−109 100 gi|7269377| ref|NP_194256.1| LN
invertase/pectin
methylesterase inhibitor
family protein
693 1.00E−57 100 gi|7340686| ref|NP_195832.1|thylakoid LN
membrane one helix protein
(OHP) [Arabidopsis
thaliana]
694 0 100 gi|21689839| gb|AAM67563.1|putative PEG
protein transport protein
SEC12p [Arabidopsis
thaliana]
695 1.00E−36 100 gi|10177015| ref|NP_199045.1|ubiquitin PEG
family protein [Arabidopsis
thaliana]
696 1.00E−106 100 gi|20466087| ref|NP_179457.1|zinc finger PEG
(C3HC4-type RING finger)
family protein
697 0 100 gi|7267203| ref|NP_192355.1|aspartyl PEG
protease family protein
[Arabidopsis thaliana]
698 0 100 gi|46931342| ref|NP_850347.1|F-box PEG
family protein [Arabidopsis
thaliana]
699 0 60 gi|46931354| gb|AAT06481.1|At3g23540 PEG
[Arabidopsis thaliana]
700 1.00E−161 100 gi|21594556| gb|AAM66021.1|plasma PEG
membrane intrinsic protein
SIMIP [Arabidopsis
thaliana]
701 0 100 gi|902923| dbj|BAA07547.1|phosphoinositide PEG
specific
phospholipase C
[Arabidopsis thaliana]
702 0 100 gi|26449713| ref|NP_195154.2|transducin PEG
family protein/WD-40
repeat family protein
703 0 94 gi|15232616| ref|NP_188176.1|phototropic- PEG
responsive NPH3 family
protein [Arabidopsis
thaliana]
704 0 100 gi|23296692| ref|NP_189177.1|formin PEG
homology 2 domain-
containing protein/FH2
domain-containing protein
[Arabidopsis thaliana]
705 0 100 gi|51013899| ref|NP_015238.1|Ydc1p PEG
[Saccharomyces cerevisiae]
gb|AAB68212.1|Lpg21p
gb|AAG22594.1|alkaline
ceramidase Ydc1p
706 0 100 gi|23297093| ref|NP_565508.1|fructose- PEG
bisphosphate aldolase,
putative [Arabidopsis
thaliana]
707 0 99 gi|6322722| ref|NP_012795.1|Pgm1p PEG
[Saccharomyces cerevisiae]
emb|CAA50895.1|
phosphoglucomutase
708 0 99 gi|10175338| ref|NP_243583.1| PEG
glutaminase [Bacillus
halodurans C-125]
709 0 94 gi|7268277| ref|NP_193265.1| PEG
cytochrome P450 family
protein [Arabidopsis
thaliana]
710 0 100 gi|6322296| sp|P38970|HAL5_YEAST PEG
Serine/threonine-protein
kinase HAL5
711 0 100 gi|17104779| ref|NP_564866.1|U-box PEG
domain-containing protein
712 0 99 gi|16080934| ref|NP_391762.1|aldehyde PEG
dehydrogenase [Bacillus
subtilis subsp. subtilis str.
168]
713 0 87 gi|37524894| ref|NP_928238.1|glutamate- PEG
1-semialdehyde 2,1-
aminomutase [Photorhabdus
luminescens subsp.
laumondii TTO1]
714 0 100 gi|16077848| ref|NP_388662.1|trehalose- PEG
6-phosphate hydrolase
[Bacillus subtilis subsp.
subtilis str. 168]
715 1.00E−81 100 gi|1922244| ref|NP_171654.1|late PEG
embryogenesis abundant
protein, putative/LEA
protein, putative
716 1.00E−112 100 gi|7268505| ref|NP_193486.1|Ras- PEG
related GTP-binding protein,
putative [Arabidopsis
thaliana]
717 3.00E−44 100 gi|21592597| ref|NP_563987.1| PEG
expressed protein
[Arabidopsis thaliana]
gb|AAF18498.1|Identical to
gb|Y10291 GAG1 protein
718 3.00E−97 44 gi|52353666| gb|AAU44232.1|hypothetical PEG
protein [Oryza sativa
(japonica cultivar-group)]
719 1.00E−175 63 gi|53850529| gb|AAC31235.1| PEG
hypothetical protein
[Arabidopsis thaliana]
gb|AAT71954.1|
720 1.00E−131 99 gi|16330417| ref|NP_441145.1|hypothetical PEG
protein sll1162
[Synechocystis sp. PCC
6803]
721 0 100 gi|17129862| ref|NP_484561.1| PEG
fructokinase [Nostoc sp.
PCC 7120]
722 0 100 gi|15293245| ref|NP_567109.1|COP9 PEG
signalosome complex
subunit 1/CSN complex
subunit 1 (CSN1)/COP11
protein (COP11)/FUSCA
protein (FUS6) [Arabidopsis
thaliana]
723 0 100 gi|20259583| ref|NP_180431.1|beta- PEG
ketoacyl-CoA synthase
family protein [Arabidopsis
thaliana]
724 0 100 gi|20465963| ref|NP_189034.1|beta- PEG
amylase, putative/1,4-
alpha-D-glucan
maltohydrolase, putative
[Arabidopsis thaliana]
725 4.00E−72 100 gi|32815911| ref|NP_201100.1|expressed PEG
protein [Arabidopsis
thaliana]
726 1.00E−179 100 gi|8953402| ref|NP_196701.1|protein PEG
kinase-related [Arabidopsis
thaliana]
727 0 100 gi|6522600| emb|CAB61965.1|1- PEG
aminocyclopropane-1-
carboxylic acid oxidase-like
protein
728 1.00E−145 85 gi|57899179| dbj|BAD87231.1|membrane PEG
protein-like [Oryza sativa
(japonica cultivar-group)]
729 0 87 gi|50909427| ref|XP_466202.1|putative PEG
DNA J domain protein
[Oryza sativa (japonica
cultivar-group)]
730 0 100 gi|53828613| ref|NP_179479.1|protein PEG
kinase family protein
[Arabidopsis thaliana]
731 1.00E−93 100 gi|23306374| ref|NP_200428.1|expressed PEG
protein [Arabidopsis
thaliana]
732 1.00E−172 100 gi|56121884| ref|NP_178191.1|major PEG
intrinsic family protein/
MIP family protein
733 0 98 gi|24762199| gb|AAN64166.1|unknown PEG
protein [Arabidopsis
thaliana]
734 0 100 gi|21230100| ref|NP_636017.1|phosphomannose PEG
isomerase/GDP-
mannose pyrophosphorylase
735 0 100 gi|10177277| dbj|BAB10630.1|glucose-6- PP
phosphate isomerase,
cytosolic [Arabidopsis
thaliana]
736 3.00E−61 100 gi|21537079| ref|NP_564835.1|expressed PP HS
protein [Arabidopsis
thaliana]
737 3.00E−91 100 gi|26452507| ref|NP_197149.1| PP PEG
dimethylmenaquinone
methyltransferase family
protein
738 0 100 gi|5734730| ref|NP_172592.1|glucose PP
transporter (STP1)
[Arabidopsis thaliana]
739 1.00E−113 100 gi|5734748| ref|NP_564017.1|integral PP SS PEG
membrane family protein
[Arabidopsis thaliana]
740 1.00E−153 100 gi|30693623| ref|NP_198871.2|expressed PP
protein [Arabidopsis
thaliana]
741 0 100 gi|56381993| ref|NP_177188.1| PP SS PEG
spermidine synthase 2
(SPDSYN2)/putrescine
aminopropyltransferase 2
742 0 100 gi|4678359| emb|CAB41169.1|cytochrome PP
P450-like protein
[Arabidopsis thaliana]
743 0 100 gi|15220055| ref|NP_173165.1|translation PP
initiation factor IF-2,
chloroplast, putative
[Arabidopsis thaliana]
744 1.00E−168 100 gi|21593654| ref|NP_181996.1|casein PP LL LN PEG
kinase II beta chain, putative
[Arabidopsis thaliana]
745 0 100 gi|9758987| dbj|BAB09497.1|chloroplast PP
nucleoid DNA-binding
protein-like [Arabidopsis
thaliana] ref|NP_199325.1|
aspartyl protease family
protein [Arabidopsis
thaliana]
746 0 100 gi|12642918| ref|NP_180133.1|protein PP SS
phosphatase 2C, putative/
PP2C, putative
747 0 100 gi|7270935| ref|NP_195661.1| PP
cytochrome P450 family
protein [Arabidopsis
thaliana]
748 1.00E−138 100 gi|21554052| gb|AAM63133.1|delta PP
tonoplast integral protein
delta-TIP [Arabidopsis
thaliana]
749 0 85 gi|28872439| ref|NP_795058.1|phosphoglycerate PP
mutase, 2,3-
bisphosphoglycerate-
independent
750 0 100 gi|25284116| pir||A95262probable formate PP PEG
dehydrogenase (EC 1.2.1.2)
alpha chain FdoG [imported] -
SinoRhizobium meliloti
(strain 1021) magaplasmid
pSymA
751 1.00E−55 100 gi|7629997| ref|NP_190945.1|late PP SS PEG
embryogenesis abundant
protein-related/LEA
protein-related
752 0 100 gi|23126243| ref|ZP_00108145.1|COG1523: PP SS
Type II secretory
pathway, pullulanase PulA
and related glycosidases
[Nostoc punctiforme PCC
73102]
753 0 100 gi|42563087| ref|NP_564975.2|CBS PP
domain-containing protein
[Arabidopsis thaliana]
754 0 100 gi|17104597| ref|NP_566716.1|protein PP SS LL
kinase, putative [Arabidopsis
thaliana]
755 0 100 gi|15810541| ref|NP_566876.3|protein PP SS
kinase family protein
[Arabidopsis thaliana]
756 3.00E−84 99 gi|16331120| ref|NP_441848.1|hypothetical PP
protein sll0359
[Synechocystis sp. PCC
6803]
757 0 99 gi|4835235| emb|CAB42913.1|putative PP
protein [Arabidopsis
thaliana] pir||T08405
hypothetical protein
F18B3.120 - Arabidopsis
thaliana
758 0 100 gi|18394553| ref|NP_564042.1|expressed PP
protein [Arabidopsis
thaliana]
759 0 99 gi|49176911| ref|NP_858035.1|mercuric PP
reductase [uncultured
bacterium]
760 0 100 gi|53794956| ref|ZP_00356067.1|COG4992: PP SS
Ornithine/acetylornithine
aminotransferase
[Chloroflexus aurantiacus]
761 0 100 gi|6714476| ref|NP_186761.1| PP LL
cystathionine gamma-
synthase, chloroplast/O-
succinylhomoserine (Thiol)-
lyase (CGS)
762 0 100 gi|9755789| ref|NP_197266.1|expressed PP SS LL LN PEG
protein [Arabidopsis
thaliana]
763 0 100 gi|15810649| pir||T48630 high affinity PP
nitrate transporter-like
protein - Arabidopsis
thaliana
764 1.00E−129 100 gi|26451800| ref|NP_850901.1| PP
expressed protein
[Arabidopsis thaliana]
765 4.00E−58 91 gi|50948565| ref|XP_483810.1|putative PP PEG
Bet1/Sft1-related SNARE
(AtBS14a) [Oryza sativa
766 0 100 gi|7630064| emb|CAB88286.1|serine/threonine- PP
specific protein
kinase-like protein
767 0 92 gi|50932239| ref|XP_475647.1|putative PP HS PEG
glutaryl-CoA dehydrogenase
768 1.00E−104 50 gi|30684716| ref|NP_196906.2|expressed PP SS
protein [Arabidopsis
thaliana]
769 2.00E−87 89 gi|50912633| ref|XP_467724.1|unknown PP PEG
protein [Oryza sativa
(japonica cultivar-group)]
770 9.00E−13 94 gi|6682226| gb|AAF23278.1|unknown PP HS SS PEG
protein [Arabidopsis
thaliana]
771 2.00E−90 49 gi|31430516| ref|NP_920131.1|putative PP
Magnaporthe grisea
pathogenicity protein
772 0 100 gi|23297753| ref|NP_192381.1|calcium- SP HS PEG
dependent protein kinase,
putative/CDPK, putative
773 3.00E−54 100 gi|4417292| ref|NP_179791.1| SP CK PP PEG
expressed protein
[Arabidopsis thaliana]
774 0 100 gi|20148423| ref|NP_177550.1| SP
sulfotransferase family
protein [Arabidopsis
thaliana]
775 4.00E−88 100 gi|21554027| ref|NP_192830.1| SP SS
transcriptional coactivator
p15 (PC4) family protein
(KELP)
776 1.00E−100 100 gi|22136806| ref|NP_176726.1|flowering SP SS PEG
locus T protein (FT)
[Arabidopsis thaliana]
777 6.00E−93 100 gi|21592464| ref|NP_565844.1|zinc finger SP LN
(AN1-like) family protein
[Arabidopsis thaliana]
778 1.00E−101 100 gi|8778645| gb|AAF79653.1|F5O11.17 SP LN
[Arabidopsis thaliana]
779 2.00E−80 100 gi|28973123| ref|NP_196373.1|glycine- SP CK
rich protein (GRP20)
[Arabidopsis thaliana]
780 0 100 gi|11994377| ref|NP_188020.1| SP PP PEG
exopolygalacturonase/
galacturan 1,4-alpha-
galacturonidase/pectinase
781 0 100 gi|20465689| ref|NP_200440.1| SP CK
peroxisomal targeting signal
type 1 receptor (PEX5)
782 8.00E−51 100 gi|21536923| ref|NP_194774.1|glycine- SP CK
rich protein [Arabidopsis
thaliana]
783 1.00E−124 100 gi|30023686| ref|NP_197608.1|leucine- SP DS PEG
rich repeat protein, putative
[Arabidopsis thaliana]
784 3.00E−76 100 gi|26453292| ref|NP_563653.2| SP SS LN
NPR1/NIM1-interacting
protein 1 (NIMIN-1)
[Arabidopsis thaliana]
785 1.00E−90 100 gi|15220022| ref|NP_173727.1|C2 SP CS PEG
domain-containing protein
[Arabidopsis thaliana]
786 2.00E−97 100 gi|56479608| ref|NP_706078.2|hypoxanthine SP SS PEG
phosphoribosyltransferase
[Shigella flexneri 2a str.
301] EDL933]
787 1.00E−130 99 gi|16331548| ref|NP_442276.1|hypothetical SP SS
protein slr0630
[Synechocystis sp. PCC
6803]
788 0 100 gi|20259167| ref|NP_568786.1|protein SP
phosphatase 2C, putative/
PP2C, putative
789 0 100 gi|6466946| ref|NP_974249.1| SP PEG
prephenate dehydratase
family protein [Arabidopsis
thaliana]
790 1.00E−130 100 gi|20465721| ref|NP_174536.1|plastid SP CS PEG
developmental protein DAG,
putative
791 1.00E−63 84 gi|10177620| dbj|BAB10767.1|unnamed SP SS
protein product [Arabidopsis
thaliana]
792 1.00E−44 100 gi|58299| emb|CAA48415.1|unnamed SP CK PEG
protein product [synthetic
construct]
793 1.00E−108 54 gi|3150411| ref|NP_180570.1|GCN5- SP CS
related N-acetyltransferase
(GNAT) family protein
794 0 100 gi|16331766| ref|NP_442494.1|aldehyde SP SS PEG
dehydrogenase
[Synechocystis sp. PCC
6803]
795 1.00E−30 100 gi|7340711| emb|CAB82954.1|cytochrome SP CK CS PEG
c oxidase subunit 5c-like
protein [Arabidopsis
thaliana]
796 1.00E−137 100 gi|5430750| ref|NP_175374.2| SP CS HS PEG
expressed protein
[Arabidopsis thaliana]
797 1.00E−93 100 gi|19310667| ref|NP_188286.1| SP PP SS
translationally controlled
tumor family protein
[Arabidopsis thaliana]
798 0 100 gi|15293235| ref|NP_194825.1|CBL- SP LN
interacting protein kinase 6
(CIPK6) [Arabidopsis
thaliana]
799 1.00E−165 62 gi|4220476| gb|AAD12699.1|putative SP
ribophorin I [Arabidopsis
thaliana]
800 0 100 gi|20908080| ref|NP_200169.1| SS
RabGAP/TBC domain-
containing protein
[Arabidopsis thaliana]
801 1.00E−68 99 gi|51449987| ref|NP_189210.2| SS PEG
myrcene/ocimene synthase,
putative [Arabidopsis
thaliana]
802 2.00E−94 100 gi|21592517| ref|NP_564266.1| SS
expressed protein
[Arabidopsis thaliana]
803 0 100 gi|42572237| ref|NP_974213.1|ankyrin SS PEG
repeat family protein/
regulator of chromosome
condensation (RCC1) family
protein [Arabidopsis
thaliana]
804 1.00E−115 100 gi|21593862| ref|NP_190632.1|kip- SS
related protein 2 (KRP2)/
cyclin-dependent kinase
inhibitor 2 (ICK2)/cdc2a-
interacting protein
[Arabidopsis thaliana]
805 0 100 gi|8843792| ref|NP_200680.1|PRLI- SS LN
interacting factor, putative
[Arabidopsis thaliana]
806 0 90 gi|21586474| gb|AAM55306.1|auxin SS
influx carrier protein
[Medicago truncatula]
807 0 99 gi|20799715| gb|Aref|NP_190468.1| SS
AMP-dependent synthetase
and ligase family protein
808 0 100 gi|15293255| ref|NP_564424.1|PHD SS
finger family protein
[Arabidopsis thaliana]
809 0 100 gi|24030409| ref|NP_194207.1| SS LN
expressed protein
[Arabidopsis thaliana]
810 0 100 gi|6728973| ref|NP_186938.1|leucine- SS PEG
rich repeat transmembrane
protein kinase, putative
811 1.00E−133 100 gi|27754697| gb|AAO22792.1|putative SS PEG
cytochrome c
oxidoreductase [Arabidopsis
thaliana]
812 0 91 gi|4539445| ref|NP_192805.1| SS
transcription elongation
factor-related [Arabidopsis
thaliana]
813 0 100 gi|23296600| ref|NP_568107.1|pseudo- SS
response regulator 7
(APRR7) [Arabidopsis
thaliana]
sp|Q93WK5|APRR7_ARATH
Two-component response
regulator-like APRR7
(Pseudo-response regulator
7)
814 0 99 gi|16331392| ref|NP_442120.1|ribulose SS
bisphosphate carboxylase
large subunit
815 0 99 gi|16329673| ref|NP_440401.1|mercuric SS
reductase [Synechocystis sp.
PCC 6803]
816 0 100 gi|23506207| gb|AAN31115.1|At2g26250/ SS
T1D16.11 [Arabidopsis
thaliana] gb|AAG60062.1|
putative beta-ketoacyl-CoA
synthase FIDDLEHEAD
817 0 99 gi|6016708| ref|NP_186798.1|protein SS
kinase, putative [Arabidopsis
thaliana]
818 0 100 gi|21593811| gb|AAM65778.1|putative SS
NADPH quinone
oxidoreductase [Arabidopsis
thaliana]
819 0 100 gi|23125809| ref|ZP_00107727.1|COG0166: SS
Glucose-6-phosphate
isomerase [Nostoc
punctiforme PCC 73102]
820 0 100 gi|16077353| ref|NP_388166.1|hypothetical SS PEG
protein BSU02840
[Bacillus subtilis subsp.
subtilis str. 168]
821 1.00E−117 100 gi|7269793| ref|NP_194624.1|Rac-like SS
GTP-binding protein
(ARAC7) [Arabidopsis
thaliana]
822 1.00E−128 96 gi|27808548| gb|AAO24554.1|At1g61150 SS PEG
[Arabidopsis thaliana]
823 0 97 gi|30684071| ref|NP_850128.1|protein SS
kinase family protein
[Arabidopsis thaliana]
824 2.00E−77 99 gi|16330866| ref|NP_441594.1|hypothetical SS LL
protein slr1273
[Synechocystis sp. PCC
6803]
825 1.00E−177 100 gi|23297050| ref|NP_194073.2|short- SS
chain
dehydrogenase/reductase
(SDR) family protein
826 1.00E−109 100 gi|12083284| ref|NP_173437.1|Rac-like SS
GTP-binding protein
(ARAC4)/Rho-like GTP-
binding protein (ROP2)
827 0 100 gi|20465939| ref|NP_850393.1|sugar SS
transporter family protein
[Arabidopsis thaliana]
828 0 98 gi|2661128| gb|AAC04613.1|arginase SS
[Glycine max] pir||T06222
probable arginase (EC
3.5.3.1) - soybean
sp|O49046|ARGI_SOYBN
Arginase
829 0 64 gi|40539114| gb|AAR87370.1|expressed SS
protein [Oryza sativa
(japonica cultivar-group)]
830 0 91 gi|52137615| emb|CAH40838.1|protein- SS
O-fucosyltransferase 1
[Saccharum officinarum]
831 0 100 gi|5733881| ref|NP_175261.1|G protein SS
coupled receptor-related
[Arabidopsis thaliana]
832 8.00E−88 100 gi|15217930| ref|NP_176128.1|hypothetical SS
protein [Arabidopsis
thaliana]
833 1.00E−29 75 gi|20465895| ref|NP_974937.1|RNA SS LN
recognition motif (RRM)-
containing protein
834 5.00E−79 100 gi|3927837| ref|NP_180456.1| SS
mitochondrial import inner
membrane translocase
subunit
Tim17/Tim22/Tim23 family
protein [Arabidopsis
thaliana]
835 1.00E−115 100 gi|21554015| ref|NP_565766.1| SS
glycolipid transfer protein-
related [Arabidopsis
thaliana]
836 1.00E−131 100 gi|26452816| ref|NP_193484.1|ubiquitin SS
carboxyl-terminal hydrolase
837 1.00E−102 99 gi|10177705| ref|NP_199436.1|inward SS LN
rectifying potassium channel
(KAT1)
838 1.00E−144 100 gi|7669950| ref|NP_190844.1|integral SS PEG
membrane Yip1 family
protein [Arabidopsis
thaliana]
839 0 100 gi|10177595| ref|NP_201509.1|protein SS LN
kinase family protein
[Arabidopsis thaliana]
840 4.00E−27 100 gi|38566508| ref|NP_200137.1| SS
arabinogalactan-protein,
putative (AGP22)
[Arabidopsis thaliana]
841 0 100 gi|18391117| ref|NP_563862.1|expressed SS PEG
protein [Arabidopsis
thaliana]
842 0 92 gi|50251322| dbj|BAD28194.1|putative SS
MFAP1 protein [Oryza
sativa (japonica cultivar-
group)]
843 1.00E−112 95 gi|3201969| gb|AAC19375.1|submergence SS
induced protein 2A [Oryza
sativa]
844 1.00E−74 88 gi|29371519| gb|AAO72703.1|unknown SS
[Oryza sativa (japonica
cultivar-group)]
845 0 100 gi|28416475| ref|NP_187911.1| SS PEG
transporter-related
[Arabidopsis thaliana]
846 0 100 gi|21323473| ref|NP_599939.1|detergent SS LN
sensitivity rescuer dtsR2
[Corynebacterium
glutamicum ATCC 13032]
847 0 82 gi|50928705| gb|AAQ14479.1|putative SS
aminotransferase [Oryza
sativa]
848 0 99 gi|3695005| gb|AAC63962.1|pyruvate SS
dehydrogenase kinase
isoform 2; PDK2 [Zea mays]
849 0 100 gi|10175838| ref|NP_244081.1| SS PEG
hypothetical protein BH3215
[Bacillus halodurans C-125]
850 0 100 gi|33589668| ref|NP_564285.1| SS PEG
calmodulin-binding protein
[Arabidopsis thaliana]

Trait Improvement Screens
DS-Improvement of drought tolerance identified by a soil drought stress tolerance screen: Drought or water deficit conditions impose mainly osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition. Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3. Such recombinant DNA may find particular use in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition can also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
PEG-Improvement of drought tolerance identified by PEG induced osmotic stress tolerance screen: Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better drought conditions providing a higher yield potential as compared to control plants.
SS-Improvement of drought tolerance identified by high salinity stress tolerance screen: Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. The present invention provides genes that help plants to maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
HS-Improvement of drought tolerance identified by heat stress tolerance screen: Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified by the present invention as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
CK and CS-Improvement of tolerance to cold stress: Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. In the present invention, two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8° C. from planting until day 28 post plating. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen can survive better cold conditions providing a higher yield potential as compared to control plants.
Improvement of tolerance to multiple stresses: Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, the plant can be enabled to react to different kinds of stresses. For examples, PEP SEQ ID NO: 459 can be used to improve both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 440 can resist heat stress, salt stress and cold stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
PP-Improvement of early plant growth and development: It has been known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” used herein encompasses the stages of seed imbibition through the early vegetative phase. The present invention provides genes that are useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, it can be recognized by those skilled in the art that genes conferring the growth advantage in early stages to plants may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. The present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 529 which can improve the plant early growth and development, and impart salt and cold tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
SP-Improvement of late plant growth and development: “Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity. In certain embodiments, transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3, exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. On one hand, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. On the other hand, the seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
LL-Improvement of tolerance to shade stress identified in a low light screen: The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. The present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by the present invention may be suitable for a higher density planting, thereby resulting increased yield per unit area.
LN-Improvement of Tolerance to Low Nitrogen Availability Stress
Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
According to the present invention, transgenic plants generated using the recombinant nucleotides, which confer enhanced nitrogen use efficiency, identified as such in Table 3, exhibit one or more desirable traits including, but not limited to, increased seedling weight, greener leaves, increased number of rosette leaves, increased or decreased root length. One skilled in the art may recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention may be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless. The transgenic plants also may be advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
Stacked Traits: The present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. patent application publications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.
Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant or different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 425, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 426 through SEQ ID NO: 850. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 851 through SEQ ID NO: 33634. In yet another aspect, the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous. Preferably, they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications. Of particular interest in the present invention are corn and soybean plants. The recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
In certain embodiments, the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart. Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures may be made on greenhouse or field grown plants and may include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
Trait data on morphological changes may be collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits may be identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain may be confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
To confirm hybrid yield in transgenic corn plants expressing genes of the present invention, it may be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein. Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture. Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.
EXAMPLES Example 1 Identification of Recombinant DNA that Confers Improved Trait(s) to Plants A. Expression Constructs for Arabidopsis Plant Transformation
Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.
B. Soil Drought Tolerance Screen
This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m2/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). Table 4 provides a list of recombinant DNA constructs that improve drought tolerance in transgenic plants.
TABLE 4
PEP Drought Seed Time to
SEQ score yield wilting
ID Construct Nomination Delta P- Delta P- Risk score P-
NO ID ID Orientation mean value mean value mean value
601 12116 CGPG1112 ANTI- 0.119 0.496 0.736 0.014 −0.026 1.000
SENSE
602 13053 CGPG1284 ANTI- 0.122 0.532 0.785 0.028 −0.122 1.000
SENSE
603 14733 CGPG1640 SENSE 0.469 0.016 −0.225 0.363 0.263 1.000
604 16132 CGPG2136 SENSE 0.149 0.529 0.472 0.046 / /
605 18276 CGPG3542 SENSE 0.320 0.088 0.433 0.264 0.362 1.000
606 70851 CGPG1691 SENSE 0.267 0.065 0.427 0.017 / /
607 70941 CGPG4067 SENSE 0.254 0.056 0.533 0.006 / /
450 71814 CGPG4434 SENSE 0.241 0.186 0.719 0.020 0.348 1.000
609 72326 CGPG27 SENSE 0.300 0.149 0.674 0.036 0.152 1.000
608 72134 CGPG5335 SENSE 0.239 0.169 0.613 0.077 0.010 1.000
783 72824 CGPG4998 SENSE 0.254 0.180 0.608 0.046 / /
610 72978 CGPG3441 SENSE 0.447 0.094 −0.973 0.071 / /
611 73619 CGPG4375 SENSE 0.990 0.022 −0.204 0.283 0.900 1.000
612 73737 CGPG5176 SENSE 0.162 0.293 1.126 0.000 0.333 1.000
460 73025 CGPG5665 SENSE 0.065 0.731 0.489 0.095 / /
613 74196 CGPG6637 SENSE 0.172 0.230 1.533 0.001 / /
614 74546 CGPG661 SENSE −0.052 0.727 0.777 0.030 −0.226 1.000
615 74558 CGPG869 SENSE 0.344 0.072 −0.138 0.117 0.033 0.955
616 74643 CGPG6159 SENSE 0.369 0.042 −1.232 0.125 0.556 1.000
617 75234 CGPG5931 SENSE 0.157 0.038 −0.095 0.479 0.026 0.881
618 75278 CGPG6282 SENSE 0.110 0.200 1.027 0.003 0.057 0.789
587 75289 CGPG6295 SENSE 0.627 0.004 −0.074 0.267 0.196 0.883
619 75585 CGPG7671 SENSE 0.474 0.008 −0.452 0.259 / /
620 76063 CGPG1574 SENSE 0.105 0.637 0.985 0.062 0.023 1.000
593 76318 CGPG8909 SENSE 0.509 0.009 1.093 0.005 0.471 0.952
599 76891 CGPG9034 SENSE 0.186 0.115 0.796 0.003 0.128 1.000
621 77401 CGPG9294 SENSE −0.072 0.643 0.554 0.061 −0.090 1.000

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
C. Heat Stress Tolerance Screen
Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature. T2 seeds were plated on ½× MS salts, 1/% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m2/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1 L. Table 5 provides a list of recombinant DNA constructs that improve heat tolerance in transgenic plants.
TABLE 5
Growth
Root stage Seedling
length at day 14 weight at
PEP at day 14 Risk day 14
seq Construct Nomination Delta P- score P- Delta P-
id ID ID Orientation mean value mean value mean value
772 10923 CGPG414 ANTI-SENSE 0.443 0.032 / / / /
525 15419 CGPG1788 ANTI-SENSE 0.385 0.040 −0.070   / 0.416 0.076
527 17903 CGPG1908 SENSE 0.125 0.091 / / / /
623 18330 CGPG3336 SENSE 0.274 0.174 1.014 0.310 1.279 0.012
624 18409 CGPG3613 SENSE 0.181 0.098 / / / /
736 18410 CGPG3614 SENSE 0.398 0.094 / / / /
432 17410 CGPG2446 SENSE 0.199 0.067 0.241 0.304 0.828 0.271
622 17808 CGPG2435 SENSE 0.266 0.040 / / / /
438 19760 CGPG4065 SENSE 0.015 0.771 −0.111   / 1.611 0.005
625 19813 CGPG4168 SENSE 0.183 0.088 0.643 0.344 0.807 0.038
440 70510 CGPG2488 SENSE 0.607 0.056 0.171 0.184 0.863 0.021
443 70812 CGPG607 SENSE 0.319 0.080 0.882 0.345 0.934 0.055
447 71446 CGPG185 SENSE 0.427 0.110 0.184 0.160 0.470 0.068
452 72005 CGPG5253 SENSE 0.209 0.098 0.331 0.586 1.178 0.122
545 72603 CGPG646 SENSE 0.476 0.029 −0.170   0.149 1.272 0.185
611 73619 CGPG4375 SENSE 0.368 0.058 0.853 0.241 0.746 0.102
467 73662 CGPG4927 SENSE 0.205 0.053 / / / /
453 72026 CGPG5231 SENSE 0.589 0.012 / / / /
626 73580 CGPG6517 SENSE −0.077 0.602 −0.195   / 1.106 0.052
628 74124 CGPG6631 SENSE 0.256 0.055 0.460 0.013 0.512 0.110
627 73935 CGPG993 SENSE 0.284 0.089 1.102 0.262 1.098 0.115
482 74707 CGPG4914 SENSE 0.077 0.680 0.099 0.745 0.773 0.058
629 74725 CGPG5393 SENSE 0.351 0.038 / / / /
591 76220 CGPG6189 SENSE 0.381 0.039 / / / /
796 75910 CGPG1256 SENSE 0.514 0.081 / / / /
630 76403 CGPG1313 SENSE 0.158 0.075 −0.033   0.864 0.252 0.581
595 76513 CGPG5892 SENSE 0.281 0.051 / / / /
631 77366 CGPG8150 SENSE 0.259 0.044 −0.116   0.058 0.354 0.369
767 77115 CGPG9135 SENSE 0.269 0.094 / / / /
770 77159 CGPG9202 SENSE 0.170 0.502 −0.014   0.887 0.389 0.048

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
D. Salt Stress Tolerance Screen
This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m2. On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1 L. Table 6 provides a list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants
TABLE 6
Root Root Growth Seedling
PEP length at length at stage weight at
SEQ day 11 day 14 at day 14 day 14
ID Construct Delta P- Delta P- RS P- Delta P-
NO ID Orientation mean value mean value mean value mean value
800 16023 SENSE 0.333 0.007 0.308 0.005 0.400 0.311 0.469 0.033
801 16424 ANTI- 0.193 0.059 0.184 0.033 0.312 0.470 0.303 0.068
SENSE
527 17903 SENSE 0.094 0.034 0.028 0.432 1.109 0.283 0.185 0.041
529 18284 SENSE 0.183 0.063 0.290 0.046 −0.043 0.739 0.584 0.100
623 18330 SENSE 0.583 0.018 0.584 0.022 0.672 0.063 1.267 0.039
531 18349 SENSE 0.191 0.046 0.218 0.107 −0.013 0.726 0.147 0.628
802 17807 SENSE 0.162 0.175 0.238 0.094 1.412 0.081 0.606 0.043
775 18214 SENSE 0.233 0.067 0.306 0.032 0.608 0.128 0.859 0.035
433 18401 SENSE 0.229 0.117 0.261 0.161 1.721 0.138 0.691 0.057
739 19624 SENSE 0.150 0.071 0.074 0.285 0.071 0.348 0.163 0.086
536 19944 SENSE 0.200 0.018 0.284 0.106 0.704 0.356 0.578 0.038
776 70236 SENSE 0.265 0.000 0.262 0.019 1.387 0.280 0.628 0.027
440 70510 SENSE 0.037 0.763 0.021 0.799 1.329 0.020 0.220 0.011
441 70605 SENSE 0.341 0.058 0.390 0.036 0.802 0.002 0.668 0.008
803 70802 SENSE 0.244 0.025 0.221 0.030 0.948 0.275 0.540 0.035
443 70812 SENSE 0.229 0.150 0.259 0.036 0.644 0.181 0.483 0.054
804 70817 SENSE 0.346 0.008 0.322 0.010 0.417 0.147 0.613 0.050
805 70819 SENSE 0.018 0.846 0.100 0.108 −0.250 0.009 0.062 0.097
806 70908 SENSE 0.284 0.070 0.327 0.028 0.955 0.158 0.935 0.002
445 70939 SENSE 0.241 0.025 0.162 0.001 1.414 0.143 0.376 0.056
607 70941 SENSE 0.648 0.008 0.656 0.002 3.713 0.006 1.061 0.009
741 71215 SENSE 0.253 0.023 0.193 0.026 2.155 0.037 0.561 0.019
447 71446 SENSE 0.288 0.014 0.312 0.014 1.490 0.088 0.794 0.011
807 72351 SENSE 0.234 0.026 0.213 0.000 1.041 0.134 0.247 0.062
545 72603 SENSE 0.688 0.007 0.570 0.007 0.854 0.070 1.029 0.028
459 72983 SENSE 0.113 0.198 0.119 0.079 1.072 0.576 0.304 0.081
453 72026 SENSE 0.222 0.149 0.255 0.054 3.706 0.006 0.715 0.053
810 73709 SENSE 0.180 0.055 0.173 0.055 0.209 0.342 0.493 0.114
557 73750 SENSE 0.184 0.037 0.168 0.021 0.111 0.585 0.573 0.002
558 73751 SENSE 0.039 0.765 0.129 0.101 1.961 0.091 0.487 0.055
786 73578 SENSE 0.194 0.004 0.103 0.008 0.330 0.137 −0.017 0.929
813 73851 SENSE 0.644 0.005 0.500 0.000 3.097 0.026 0.955 0.005
787 74112 SENSE 0.390 0.007 0.257 0.001 0.636 0.178 0.337 0.156
814 74156 SENSE 0.395 0.001 0.328 0.004 0.829 0.079 0.454 0.000
815 74194 SENSE 0.062 0.352 0.057 0.218 0.995 0.082 0.173 0.040
562 73929 SENSE 0.211 0.311 0.245 0.125 0.822 0.111 0.621 0.071
816 74252 SENSE 0.678 0.010 0.604 0.018 1.627 0.027 1.330 0.028
569 74315 SENSE −0.087 0.327 −0.042 0.472 0.088 0.600 0.282 0.094
817 74317 SENSE 0.227 0.082 0.205 0.122 3.190 0.059 0.424 0.011
476 74388 SENSE 0.185 0.112 0.140 0.084 1.750 0.110 0.533 0.032
819 74506 SENSE 0.211 0.032 0.220 0.038 1.329 0.203 0.525 0.016
479 74522 SENSE 0.262 0.022 0.188 0.076 0.562 0.265 0.184 0.494
820 74540 SENSE 0.318 0.066 0.219 0.094 0.137 0.610 0.299 0.316
752 74577 SENSE 0.216 0.144 0.183 0.041 1.105 0.296 0.518 0.020
808 73087 SENSE 0.125 0.466 0.200 0.059 0.067 0.398 0.446 0.043
818 74328 SENSE 0.069 0.309 0.092 0.005 0.544 0.442 0.354 0.000
821 74650 SENSE 0.015 0.657 0.008 0.919 1.307 0.031 0.352 0.026
614 74546 SENSE 0.318 0.003 0.307 0.001 1.709 0.071 0.524 0.005
824 74956 SENSE 0.194 0.005 0.199 0.064 1.553 0.063 0.455 0.024
751 74556 SENSE 0.006 0.952 0.109 0.212 0.334 0.283 0.419 0.080
791 74393 SENSE 0.410 0.023 0.384 0.057 0.656 0.002 0.696 0.020
826 75255 SENSE −0.002 0.984 0.033 0.689 0.518 0.504 0.490 0.093
827 75269 SENSE 0.122 0.095 0.095 0.278 1.520 0.292 0.519 0.046
587 75289 SENSE 0.218 0.001 0.213 0.024 2.926 0.112 0.543 0.021
809 73223 SENSE 0.340 0.140 0.249 0.179 0.420 0.351 0.729 0.073
784 73283 SENSE 0.128 0.292 0.320 0.200 −0.006 0.682 0.032
811 73804 SENSE 0.174 0.069 0.090 0.328 0.140 0.536 0.095 0.609
559 73814 SENSE 0.135 0.100 0.185 0.082 0.800 0.293 0.375 0.104
812 73819 SENSE 0.344 0.017 0.321 0.021 2.159 0.117 0.672 0.027
471 73863 SENSE 0.335 0.030 0.251 0.041 1.905 0.220 0.563 0.050
472 74059 SENSE 0.265 0.039 0.293 0.022 −0.146 0.035 0.298 0.161
746 74080 SENSE 0.107 0.509 0.130 0.133 1.101 0.293 0.584 0.071
482 74707 SENSE 0.544 0.029 0.623 0.013 0.966 0.104 1.122 0.007
822 74718 SENSE 0.289 0.111 0.382 0.021 0.648 0.404 0.646 0.112
580 74733 SENSE 0.152 0.138 0.129 0.095 0.190 0.685 0.220 0.338
823 74745 SENSE 0.166 0.035 0.123 0.240 1.102 0.100 0.580 0.014
581 74749 SENSE 0.444 0.048 0.478 0.010 0.796 0.173 1.076 0.008
582 74752 SENSE 0.260 0.310 0.450 0.088 0.467 0.284 0.947 0.022
754 74753 SENSE −0.058 0.235 0.082 0.326 0.330 0.005
755 74754 SENSE 0.230 0.088 0.228 0.077 0.966 0.353 0.407 0.118
484 74755 SENSE 0.070 0.574 −0.020 0.470 0.265 0.208 0.455 0.073
493 75355 SENSE 0.290 0.057 0.305 0.000 0.440 0.425 0.360 0.301
828 75432 SENSE 0.249 0.020 0.178 0.100 0.475 0.196 0.216 0.454
829 75468 SENSE 0.146 0.344 0.185 0.054 1.373 0.268 0.311 0.141
794 75752 SENSE 0.402 0.058 0.394 0.049 0.521 0.335 0.672 0.027
760 75931 SENSE 0.104 0.239 0.074 0.433 1.121 0.209 0.321 0.069
832 76064 SENSE 0.349 0.011 0.270 0.046 0.152 0.360 0.250 0.295
797 76101 SENSE 0.527 0.064 0.542 0.107 0.724 0.367 1.480 0.024
833 76121 SENSE −0.026 0.849 0.019 0.829 2.841 0.059 0.069 0.822
834 76193 SENSE 0.262 0.020 0.254 0.021 0.834 0.097 0.441 0.054
835 76237 SENSE 0.334 0.098 0.365 0.047 0.727 0.042 0.786 0.137
836 76281 SENSE 0.167 0.154 0.181 0.184 0.572 0.096 0.598 0.092
762 76293 SENSE 0.042 0.763 −0.044 0.776 0.769 0.270 0.610 0.017
831 75911 SENSE 0.179 0.041 0.143 0.083 1.508 0.266 0.464 0.001
830 75686 SENSE 0.378 0.036 0.358 0.039 1.039 0.057 0.666 0.014
837 76388 SENSE 0.209 0.054 0.163 0.136 0.853 0.367 0.485 0.013
838 76557 SENSE 0.165 0.042 0.104 0.095 1.943 0.027 0.395 0.031
839 76567 SENSE −0.046 0.551 0.067 0.196 1.685 0.108 0.273 0.053
507 76575 SENSE 0.101 0.302 0.183 0.067 0.121 0.496 0.298 0.306
840 76624 SENSE 0.399 0.047 0.367 0.041 0.652 0.122 0.622 0.069
597 76719 SENSE 0.229 0.051 0.140 0.317 0.726 0.330 0.487 0.083
841 76764 SENSE 0.482 0.036 0.376 0.036 2.259 0.163 0.670 0.127
825 75076 SENSE 0.186 0.139 0.233 0.016 2.197 0.062 0.538 0.031
843 76976 SENSE 0.455 0.013 0.375 0.044 3.499 0.020 0.737 0.007
844 76985 SENSE 0.313 0.050 0.299 0.037 3.310 0.041 0.587 0.048
842 76855 SENSE 0.463 0.026 0.636 0.010 0.188 0.020 1.334 0.001
599 76891 SENSE 0.325 0.000 0.368 0.003 0.445 0.087 0.795 0.003
845 77014 SENSE 0.005 0.889 −0.040 0.597 0.024 0.932 0.265 0.098
522 77351 SENSE 0.392 0.048 0.454 0.014 2.912 0.052 1.340 0.006
631 77366 SENSE 0.214 0.007 0.159 0.017 0.205 0.076 0.382 0.029
846 77112 SENSE −0.001 0.986 0.020 0.801 1.640 0.020 0.583 0.025
847 77117 SENSE 0.219 0.058 0.234 0.116 1.406 0.172 0.516 0.072
768 77128 SENSE 0.239 0.020 0.312 0.019 0.324 0.523 0.470 0.028
520 77135 SENSE 0.153 0.070 0.133 0.059 0.386 0.503 0.344 0.064
848 77138 SENSE 0.171 0.030 0.271 0.012 1.211 0.256 0.482 0.035
770 77159 SENSE 0.188 0.115 0.220 0.110 1.176 0.381 0.589 0.039
600 77208 SENSE 0.321 0.075 0.254 0.154 0.183 0.578 0.540 0.059
521 77219 SENSE 0.297 0.090 0.413 0.018 2.299 0.024 1.010 0.014
849 77226 SENSE 0.164 0.193 0.098 0.413 1.279 0.142 0.264 0.043
621 77401 SENSE 0.317 0.068 0.274 0.018 1.134 0.258 0.502 0.213
850 77418 SENSE 0.204 0.096 0.201 0.110 0.344 0.184 0.626 0.005

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen
There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.
T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½× MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1 L. Table 7 provides a list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants.
TABLE 7
Root Root Growth Seedling
PEP length at length at stage weight
SEQ day 11 day 14 at day 14 at day 14
ID Construct Delta P- Delta P- RS P- Delta P-
NO ID Orientation mean value mean value mean value mean value
694 10443 ANTI- 0.162 0.251 0.127 0.118 3.475 0.022 0.547 0.157
SENSE
772 10923 ANTI- 0.235 0.033 0.332 0.010 3.496 0.020 0.284 0.098
SENSE
524 14835 ANTI- 0.195 0.134 0.126 0.173 1.477 0.362 0.300 0.050
SENSE
773 14836 SENSE 0.210 0.159 0.037 0.707 2.867 0.040 0.205 0.332
801 16424 ANTI- 0.309 0.055 0.201 0.210 2.261 0.150 0.375 0.012
SENSE
530 18332 SENSE 0.129 0.095 0.141 0.028 2.205 0.134 0.062 0.379
737 18536 SENSE 0.214 0.036 0.228 0.065 3.062 0.082 0.323 0.153
696 18537 SENSE 0.206 0.030 0.197 0.034 2.641 0.076 0.399 0.112
697 18831 SENSE 0.002 0.988 0.030 0.828 3.243 0.050 −0.170 0.134
698 19044 ANTI- 0.300 0.082 0.220 0.006 2.465 0.113 0.284 0.113
SENSE
695 17814 SENSE 0.215 0.056 0.057 0.542 4.000 0.000 0.573 0.004
433 18401 SENSE 0.229 0.017 0.143 0.252 1.670 0.065 0.503 0.004
532 18623 SENSE 0.246 0.028 0.205 0.050 1.836 0.002 0.356 0.009
739 19624 SENSE 0.088 0.232 −0.011 0.867 1.317 0.075 0.458 0.135
699 19950 SENSE 0.225 0.119 0.214 0.072 3.393 0.030 0.335 0.078
700 70221 SENSE 0.148 0.179 0.076 0.152 3.183 0.060 0.351 0.088
776 70236 SENSE 0.094 0.489 0.115 0.414 2.368 0.033 0.116 0.179
441 70605 SENSE 0.423 0.020 0.355 0.042 3.087 0.021 0.588 0.075
803 70802 SENSE 0.182 0.345 0.089 0.596 3.228 0.053 0.183 0.477
780 70804 SENSE 0.244 0.053 0.108 0.207 2.514 0.233 0.434 0.011
539 70830 SENSE 0.226 0.030 0.064 0.387 0.914 0.354 0.272 0.028
445 70939 SENSE 0.086 0.396 −0.040 0.508 3.390 0.031 0.335 0.058
701 71110 SENSE −0.206 0.153 −0.164 0.212 1.410 0.018 −0.318 0.079
741 71215 SENSE 0.337 0.025 0.152 0.047 2.979 0.000 0.566 0.032
447 71446 SENSE 0.263 0.003 0.129 0.155 2.672 0.057 0.500 0.011
702 71718 SENSE 0.197 0.031 0.257 0.036 3.310 0.041 0.189 0.137
703 71838 SENSE 0.234 0.039 0.260 0.016 3.164 0.063 0.079 0.583
704 72087 SENSE 0.242 0.052 0.216 0.060 4.000 0.000 0.299 0.177
543 72352 SENSE 0.194 0.185 0.259 0.053 1.666 0.069 0.227 0.080
544 72413 SENSE 0.082 0.241 0.158 0.024 3.196 0.058 −0.040 0.748
545 72603 SENSE 0.178 0.197 0.180 0.208 3.093 0.076 0.127 0.489
455 72637 SENSE 0.247 0.002 0.197 0.017 3.274 0.046 0.348 0.041
546 72676 SENSE 0.141 0.015 0.181 0.094 4.000 0.000 0.301 0.017
705 72729 SENSE 0.402 0.017 0.242 0.001 2.119 0.090 0.568 0.005
783 72824 SENSE 0.166 0.281 0.349 0.071 2.355 0.107 0.278 0.212
610 72978 SENSE 0.190 0.112 0.304 0.032 1.328 0.494 −0.047 0.654
706 73302 SENSE 0.400 0.001 0.346 0.026 3.489 0.021 0.497 0.010
744 73666 SENSE 0.361 0.017 0.177 0.258 3.488 0.021 0.757 0.008
810 73709 SENSE 0.222 0.041 0.130 0.258 2.104 0.106 0.426 0.009
709 73736 SENSE 0.275 0.026 0.210 0.006 2.536 0.115 0.378 0.112
551 73031 SENSE 0.095 0.109 0.201 0.033 3.122 0.019 0.178 0.262
707 73484 SENSE 0.365 0.104 0.185 0.315 2.980 0.029 0.489 0.017
786 73578 SENSE 0.113 0.118 0.125 0.209 2.516 0.083 0.156 0.251
708 73594 SENSE 0.087 0.625 0.183 0.201 1.230 0.041 0.140 0.232
627 73935 SENSE 0.378 0.000 0.261 0.037 3.484 0.021 0.489 0.023
711 74266 SENSE −0.005 0.943 −0.050 0.648 2.571 0.074 0.287 0.074
789 74278 SENSE 0.145 0.299 0.134 0.452 3.666 0.008 0.350 0.140
790 74359 SENSE 0.365 0.026 0.291 0.112 2.220 0.053 0.351 0.030
792 74414 SENSE 0.310 0.035 0.272 0.054 2.515 0.037 0.323 0.015
479 74522 SENSE 0.224 0.077 0.169 0.016 2.547 0.086 0.178 0.333
750 74539 SENSE 0.327 0.102 0.197 0.371 3.356 0.012 0.496 0.011
820 74540 SENSE 0.290 0.007 0.376 0.013 3.350 0.036 0.458 0.096
714 74574 SENSE 0.236 0.096 0.155 0.053 2.448 0.255 0.179 0.475
715 74607 SENSE 0.274 0.007 0.285 0.004 2.783 0.053 0.341 0.007
716 74642 SENSE 0.386 0.022 0.273 0.089 2.308 0.009 0.378 0.141
615 74558 SENSE 0.184 0.012 0.053 0.528 2.052 0.036 0.097 0.259
751 74556 SENSE 0.258 0.186 0.160 0.362 2.253 0.042 0.477 0.059
488 75231 SENSE 0.035 0.555 0.061 0.577 3.350 0.036 0.253 0.059
617 75234 SENSE 0.053 0.310 0.018 0.240 3.273 0.046 0.197 0.096
587 75289 SENSE 0.226 0.032 0.245 0.019 3.458 0.024 0.506 0.052
785 73292 SENSE −0.003 0.956 0.008 0.746 2.088 0.079 0.274 0.120
811 73804 SENSE 0.305 0.051 0.273 0.170 2.250 0.150 0.377 0.021
717 75015 SENSE −0.036 0.360 −0.065 0.167 3.362 0.034 0.111 0.227
710 74016 SENSE 0.176 0.019 0.207 0.007 2.898 0.035 0.255 0.133
564 74072 SENSE 0.025 0.522 0.027 0.699 3.304 0.042 0.109 0.274
712 74479 SENSE 0.059 0.362 0.152 0.184 2.226 0.130 0.226 0.076
571 74490 SENSE 0.002 0.956 0.097 0.405 3.332 0.038 0.268 0.136
713 74549 SENSE 0.152 0.027 0.069 0.254 3.392 0.031 0.244 0.056
482 74707 SENSE 0.322 0.002 0.148 0.040 2.228 0.164 0.392 0.021
822 74718 SENSE 0.248 0.176 0.188 0.320 3.180 0.061 0.630 0.091
580 74733 SENSE 0.207 0.021 0.216 0.036 4.000 0.000 0.156 0.062
582 74752 SENSE −0.020 0.679 −0.035 0.616 1.181 0.182 0.188 0.054
484 74755 SENSE 0.517 0.002 0.386 0.016 4.000 0.000 0.686 0.005
718 75380 SENSE 0.038 0.789 −0.045 0.789 3.336 0.037 0.135 0.401
720 75765 SENSE 0.256 0.182 0.165 0.185 2.481 0.244 0.445 0.035
795 75867 SENSE −0.430 0.150 −0.192 0.321 0.708 0.082 −0.315 0.193
719 75560 SENSE 0.089 0.012 0.107 0.281 1.805 0.144 0.125 0.416
794 75752 SENSE 0.236 0.022 0.217 0.003 2.872 0.036 0.332 0.017
721 75977 SENSE 0.148 0.019 0.169 0.012 3.354 0.035 0.069 0.615
499 75984 SENSE 0.389 0.049 0.237 0.199 2.581 0.098 0.501 0.064
722 76052 SENSE 0.133 0.135 0.095 0.201 3.291 0.043 0.274 0.266
642 76061 SENSE 0.006 0.945 0.032 0.601 1.860 0.002 0.036 0.606
723 76109 SENSE 0.284 0.026 0.162 0.082 2.427 0.263 0.481 0.009
724 76116 SENSE 0.264 0.066 0.218 0.018 1.973 0.220 0.304 0.309
725 76158 SENSE 0.230 0.016 0.279 0.051 2.217 0.160 −0.038 0.034
726 76232 SENSE 0.170 0.133 0.159 0.071 1.626 0.203 0.294 0.060
727 76269 SENSE 0.281 0.030 0.304 0.034 3.131 0.069 0.243 0.096
496 75907 SENSE 0.196 0.063 0.063 0.324 0.875 0.462 0.259 0.122
796 75910 SENSE 0.196 0.135 0.246 0.025 2.591 0.069 0.357 0.009
641 75975 SENSE 0.285 0.114 0.307 0.256 3.032 0.088 0.485 0.063
762 76293 SENSE 0.212 0.059 −0.018 0.602 3.456 0.024 0.525 0.053
728 76303 SENSE −0.023 0.677 0.168 0.007 2.556 0.079 0.076 0.171
729 76474 SENSE 0.286 0.038 0.277 0.046 3.590 0.013 0.424 0.047
630 76403 SENSE 0.163 0.047 0.110 0.085 2.768 0.154 0.185 0.318
730 76529 SENSE 0.232 0.213 0.382 0.040 2.989 0.098 −0.084 0.591
838 76557 SENSE 0.056 0.461 0.046 0.675 3.376 0.033 0.249 0.045
595 76513 SENSE 0.120 0.072 0.126 0.062 1.867 0.292 0.035 0.463
731 76625 SENSE 0.091 0.042 0.123 0.011 3.184 0.060 0.200 0.024
509 76741 SENSE −0.022 0.760 0.226 0.018 2.136 0.002 −0.231 0.026
732 76759 SENSE −0.105 0.298 −0.115 0.233 2.647 0.060 −0.037 0.672
841 76764 SENSE 0.082 0.410 −0.022 0.862 4.000 0.000 0.230 0.045
514 76917 SENSE 0.305 0.073 0.275 0.066 3.294 0.043 0.387 0.027
765 76804 SENSE 0.123 0.131 0.072 0.462 2.766 0.046 0.468 0.028
599 76891 SENSE 0.377 0.014 0.318 0.058 4.000 0.000 0.676 0.036
845 77014 SENSE 0.010 0.673 0.099 0.050 2.138 0.175 −0.063 0.668
733 77026 SENSE 0.458 0.024 0.329 0.003 1.410 0.109 0.752 0.031
685 77055 SENSE 0.636 0.026 0.512 0.016 4.000 0.000 0.536 0.061
522 77351 SENSE 0.137 0.055 0.116 0.023 2.509 0.096 0.738 0.009
767 77115 SENSE 0.301 0.032 0.234 0.062 4.000 0.000 0.855 0.005
734 77131 SENSE 0.251 0.081 0.211 0.005 1.240 0.394 0.169 0.340
769 77158 SENSE 0.258 0.149 0.223 0.193 4.000 0.000 0.519 0.045
770 77159 SENSE 0.054 0.713 −0.101 0.107 3.028 0.089 0.481 0.112
521 77219 SENSE 0.187 0.242 0.207 0.103 1.728 0.268 0.419 0.022
849 77226 SENSE 0.353 0.033 0.373 0.016 4.000 0.000 0.542 0.022
621 77401 SENSE 0.264 0.045 0.174 0.196 3.288 0.044 0.404 0.003
850 77418 SENSE 0.210 0.089 0.132 0.222 3.024 0.090 0.440 0.027

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference.
If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
F. Cold Shock Tolerance Screen
This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½× Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. Table 8 provides a list of recombinant nucleotides that improve cold shock stress tolerance in plants.
TABLE 8
Rosette
Rosette area at Rosette
PEP area at day 28 area
SEQ day 8 Risk difference
ID Construct Nomination Delta P- score P- Delta
NO ID ID Orientation mean value mean value mean P-value
426 10919 CGPG699 ANTI- 0.437 0.036 0.247 0.108 0.265 0.013
SENSE
428 11310 CGPG267 ANTI- 0.224 0.560 0.497 0.218 0.832 0.055
SENSE
429 11866 CGPG1179 ANTI- 0.354 0.018 0.422 0.090 0.428 0.073
SENSE
430 11937 CGPG959 ANTI- −0.510 0.004 −0.010 0.939 0.331 0.048
SENSE
773 14836 CGPG1072 SENSE 0.862 0.055 0.187 0.379 0.147 0.611
432 17410 CGPG2446 SENSE 0.282 0.076 0.397 0.170 0.536 0.246
433 18401 CGPG1862 SENSE 0.134 0.597 0.801 0.008 0.582 0.009
434 18665 CGPG3014 SENSE 1.273 0.026 1.142 0.003 1.215 0.004
427 11142 CGPG567 SENSE 0.916 0.053 0.255 0.234 0.048 0.792
431 16218 CGPG2158 SENSE 0.443 0.278 0.475 0.081 0.501 0.074
435 19141 CGPG1674 SENSE 0.901 0.002 0.935 0.000 0.956 0.000
436 19156 CGPG2680 SENSE −0.483 0.002 0.610 0.002 0.576 0.052
437 19621 CGPG3577 SENSE 0.231 0.388 1.265 0.058 1.486 0.059
438 19760 CGPG4065 SENSE 0.759 0.043 0.833 0.029 0.983 0.021
439 19857 CGPG3929 SENSE 0.060 0.931 0.766 0.090 0.853 0.097
440 70510 CGPG2488 SENSE 0.591 0.010 0.146 0.520 −0.009 0.978
441 70605 CGPG3012 SENSE 0.442 0.088 −0.167 0.612 −0.624 0.107
442 70607 CGPG3162 SENSE 0.747 0.209 1.688 0.001 1.929 0.001
443 70812 CGPG607 SENSE 0.767 0.126 0.823 0.091 1.442 0.149
781 70821 CGPG345 SENSE 0.523 0.039 0.380 0.019 0.584 0.070
444 70933 CGPG4084 SENSE 0.506 0.071 1.352 0.009 1.660 0.012
445 70939 CGPG3917 SENSE 0.310 0.057 1.473 0.012 1.787 0.014
779 70674 CGPG4492 SENSE 0.060 0.625 1.600 0.013 1.904 0.012
446 71319 CGPG4414 SENSE 0.292 0.299 0.516 0.089 0.494 0.090
447 71446 CGPG185 SENSE 0.740 0.040 0.508 0.034 0.304 0.053
448 71609 CGPG1679 SENSE 0.373 0.408 1.184 0.076 1.318 0.073
449 71649 CGPG271 SENSE 0.652 0.037 0.656 0.157 1.012 0.060
450 71814 CGPG4434 SENSE 0.311 0.066 0.548 0.006 0.498 0.008
451 71945 CGPG2198 SENSE −0.140 0.232 0.603 0.013 0.755 0.011
452 72005 CGPG5253 SENSE 0.369 0.180 1.152 0.012 1.410 0.015
782 72505 CGPG4786 SENSE 0.487 0.021 0.632 0.086 0.714 0.077
454 72533 CGPG4815 SENSE 0.099 0.817 0.286 0.087 0.158 0.077
455 72637 CGPG4859 SENSE −0.231 0.461 0.641 0.034 0.698 0.025
456 72782 CGPG1589 SENSE 1.087 0.013 0.655 0.050 0.274 0.208
457 72794 CGPG1826 SENSE 0.375 0.150 0.648 0.076 0.528 0.074
458 72919 CGPG3899 SENSE 0.628 0.039 −0.151 0.385 −0.346 0.155
459 72983 CGPG5610 SENSE 0.760 0.006 −0.077 0.605 −0.453 0.195
463 73338 CGPG4862 SENSE 0.473 0.301 0.600 0.053 0.833 0.039
464 73344 CGPG4910 SENSE 0.350 0.074 0.394 0.170 0.280 0.453
466 73644 CGPG1756 SENSE 0.637 0.050 0.893 0.069 0.968 0.007
467 73662 CGPG4927 SENSE 0.402 0.133 1.524 0.005 1.791 0.002
468 73672 CGPG5106 SENSE 0.943 0.012 0.901 0.038 0.991 0.033
453 72026 CGPG5231 SENSE 0.447 0.104 0.699 0.124 0.705 0.084
469 73733 CGPG5167 SENSE 0.308 0.336 0.295 0.015 0.197 0.108
460 73025 CGPG5665 SENSE −0.139 0.244 0.441 0.012 0.585 0.004
462 73092 CGPG5695 SENSE 0.183 0.396 1.011 0.035 0.986 0.069
465 73536 CGPG6541 SENSE 0.767 0.043 0.731 0.024 0.826 0.024
473 74205 CGPG5089 SENSE 0.443 0.198 0.824 0.078 0.728 0.159
474 74321 CGPG5870 SENSE 0.132 0.257 0.715 0.002 0.789 0.000
475 74337 CGPG5888 SENSE 1.176 0.010 0.981 0.003 0.987 0.001
476 74388 CGPG1461 SENSE 0.489 0.116 0.480 0.092 0.297 0.287
792 74414 CGPG6664 SENSE 1.118 0.037 0.557 0.021 0.388 0.040
479 74522 CGPG82 SENSE 0.928 0.013 1.382 0.018 1.327 0.019
480 74526 CGPG6761 SENSE 0.315 0.422 0.679 0.020 0.604 0.013
481 74576 CGPG6781 SENSE 0.620 0.012 −0.164 0.143 −0.410 0.032
461 73050 CGPG5697 SENSE 0.646 0.212 1.284 0.023 1.751 0.010
485 75202 CGPG1743 SENSE 0.413 0.012 0.422 0.007 0.292 0.026
486 75220 CGPG5434 SENSE 0.791 0.037 0.778 0.015 0.787 0.020
487 75226 CGPG5824 SENSE 0.788 0.007 0.595 0.119 0.619 0.173
488 75231 CGPG5879 SENSE 0.976 0.017 0.663 0.004 0.496 0.029
489 75239 CGPG5949 SENSE 1.048 0.011 0.570 0.242 0.547 0.290
490 75258 CGPG6096 SENSE 0.808 0.047 0.435 0.135 0.585 0.025
491 75270 CGPG6218 SENSE 0.526 0.011 0.746 0.141 0.866 0.193
492 75271 CGPG6226 SENSE 0.092 0.698 0.963 0.083 0.982 0.128
470 73846 CGPG1924 SENSE 0.807 0.032 0.175 0.623 0.014 0.973
471 73863 CGPG5201 SENSE 0.285 0.404 0.740 0.021 0.797 0.016
472 74059 CGPG1884 SENSE 0.364 0.014 0.162 0.137 −0.131 0.424
477 74412 CGPG6743 SENSE 0.163 0.522 0.670 0.026 0.626 0.131
478 74445 CGPG6722 SENSE 0.805 0.065 0.361 0.164 0.584 0.145
482 74707 CGPG4914 SENSE 0.584 0.005 0.453 0.248 0.239 0.415
483 74743 CGPG5840 SENSE 0.187 0.245 0.843 0.040 0.976 0.045
484 74755 CGPG5980 SENSE 0.959 0.083 0.680 0.205 0.768 0.206
493 75355 CGPG7518 SENSE 0.268 0.006 0.715 0.053 0.799 0.038
494 75460 CGPG7654 SENSE 0.214 0.120 0.649 0.053 0.713 0.094
795 75867 CGPG6965 SENSE 0.817 0.040 0.707 0.058 0.588 0.128
497 75927 CGPG8229 SENSE 0.360 0.163 0.808 0.017 0.955 0.007
498 75962 CGPG8224 SENSE −0.337 0.167 0.398 0.061 0.706 0.030
499 75984 CGPG1927 SENSE 1.141 0.050 0.220 0.073 0.076 0.202
500 76119 CGPG5962 SENSE 0.206 0.445 1.056 0.011 1.364 0.015
501 76209 CGPG5375 SENSE 0.598 0.078 0.737 0.112 0.834 0.075
496 75907 CGPG8259 SENSE 0.730 0.002 0.376 0.023 0.494 0.065
495 75847 CGPG6875 SENSE 0.568 0.129 0.927 0.025 1.062 0.033
502 76323 CGPG8949 SENSE 0.592 0.042 0.736 0.049 0.844 0.048
503 76346 CGPG8943 SENSE 0.150 0.647 0.588 0.061 0.656 0.044
504 76352 CGPG8896 SENSE 0.464 0.551 1.124 0.109 1.375 0.049
505 76360 CGPG8960 SENSE 0.442 0.274 0.646 0.072 0.830 0.038
507 76575 CGPG7260 SENSE 0.444 0.030 0.813 0.023 1.123 0.050
506 76512 CGPG5891 SENSE −0.060 0.669 0.486 0.005 0.799 0.014
508 76733 CGPG2647 SENSE 0.304 0.312 1.028 0.019 0.976 0.034
509 76741 CGPG6995 SENSE 1.007 0.040 0.344 0.008 0.302 0.033
512 76902 CGPG9083 SENSE 0.343 0.097 0.551 0.034 0.496 0.082
513 76913 CGPG9076 SENSE 0.524 0.021 0.257 0.530 0.149 0.719
514 76917 CGPG9108 SENSE 0.728 0.063 0.804 0.015 0.779 0.036
515 76929 CGPG9109 SENSE 0.335 0.220 0.628 0.031 0.844 0.027
510 76845 CGPG9046 SENSE 0.123 0.751 0.877 0.081 1.232 0.027
511 76857 CGPG9047 SENSE 1.055 0.053 1.170 0.023 1.286 0.020
516 77009 CGPG5933 SENSE 0.604 0.051 0.853 0.104 0.864 0.120
517 77022 CGPG6335 SENSE 0.301 0.356 0.444 0.024 0.554 0.001
522 77351 CGPG8087 SENSE 0.749 0.023 1.023 0.012 1.031 0.032
518 77108 CGPG9174 SENSE 0.651 0.025 0.788 0.020 0.723 0.133
519 77125 CGPG9120 SENSE 0.945 0.049 1.641 0.013 1.794 0.011
520 77135 CGPG9200 SENSE 0.648 0.253 1.299 0.019 1.486 0.034
521 77219 CGPG9263 SENSE 0.636 0.005 0.300 0.355 0.763 0.003

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
G. Cold Germination Tolerance Screen
This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions. T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½× Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m2/s. At 28 days post plating, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1 L. Table 9 provides a list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants.
TABLE 9
Root Growth
length at stage at
PEP day 28 day28
SEQ Construct Delta Delta
ID ID Orientation mean P-value mean P-value
523 14810 SENSE 0.429 0.028 2.822 0.041
524 14835 ANTI-SENSE 0.307 0.114 2.658 0.058
525 15419 ANTI-SENSE / / 3.333 0.038
526 16223 SENSE 0.321 0.062 4.000 0.000
527 17903 SENSE / / 2.667 0.057
528 18279 SENSE 0.100 0.622 3.390 0.031
529 18284 SENSE 0.343 0.143 1.533 0.097
530 18332 SENSE 0.286 0.032 2.579 0.026
531 18349 SENSE 0.153 0.013 1.694 0.026
537 70125 SENSE 0.550 0.020 2.987 0.034
432 17410 SENSE 0.346 0.027 4.000 0.000
532 18623 SENSE 0.253 0.048 2.144 0.011
533 19551 SENSE 0.109 0.055 0.511 0.479
534 19627 SENSE 0.234 0.007 0.918 0.240
438 19760 SENSE 0.275 0.078 2.249 0.141
535 19785 SENSE 0.175 0.301 3.103 0.074
536 19944 SENSE 0.107 0.214 3.423 0.027
538 70504 SENSE −0.204 0.122 2.893 0.035
441 70605 SENSE −0.135 0.357 3.322 0.039
539 70830 SENSE 0.085 0.303 3.396 0.030
444 70933 SENSE 0.191 0.084 3.371 0.033
540 70943 SENSE 0.181 0.011 2.958 0.032
541 71711 ANTI-SENSE 0.086 0.492 3.345 0.036
542 71962 SENSE 0.251 0.096 3.025 0.090
543 72352 SENSE 0.267 0.200 3.358 0.035
544 72413 SENSE 0.316 0.087 4.000 0.000
545 72603 SENSE 0.703 0.015 3.298 0.011
546 72676 SENSE 0.164 0.097 2.360 0.012
547 72737 SENSE 0.153 0.127 2.900 0.035
548 72742 SENSE 0.320 0.038 2.868 0.127
549 72762 SENSE 0.904 0.015 2.647 0.087
552 73091 SENSE 0.357 0.027 2.858 0.038
554 73340 SENSE 0.305 0.015 1.663 0.346
453 72026 SENSE 0.365 0.027 3.359 0.035
557 73750 SENSE 0.353 0.032 4.000 0.000
558 73751 SENSE 0.668 0.008 4.000 0.000
551 73031 SENSE 0.265 0.005 4.000 0.000
555 73420 SENSE −0.007 0.910 2.116 0.031
556 73489 SENSE 0.266 0.069 3.297 0.043
567 74135 SENSE 0.092 0.134 1.544 0.014
550 72993 SENSE 0.305 0.085 2.926 0.032
562 73929 SENSE 0.260 0.018 4.000 0.000
568 74313 SENSE 0.157 0.026 3.280 0.045
569 74315 SENSE 0.158 0.012 4.000 0.000
790 74359 SENSE 0.341 0.080 2.062 0.101
570 74427 SENSE 0.132 0.097 1.952 0.051
573 74531 SENSE 0.242 0.018 4.000 0.000
574 74532 SENSE 0.288 0.079 3.361 0.034
575 74538 SENSE 0.250 0.226 3.737 0.005
576 74550 SENSE 0.245 0.043 4.000 0.000
577 74579 SENSE 0.255 0.002 3.346 0.036
578 74644 SENSE 0.105 0.492 2.711 0.052
584 74896 SENSE 0.381 0.036 4.000 0.000
585 74976 SENSE 0.134 0.038 2.342 0.293
586 74991 SENSE −0.132 0.117 1.739 0.090
488 75231 SENSE 0.042 0.074 4.000 0.000
587 75289 SENSE 0.173 0.053 4.000 0.000
553 73224 SENSE 0.245 0.077 1.439 0.015
785 73292 SENSE 0.169 0.363 2.790 0.055
559 73814 SENSE 0.305 0.075 1.976 0.058
560 73855 SENSE 0.637 0.034 3.222 0.015
561 73856 SENSE 0.650 0.011 2.895 0.056
471 73863 SENSE 0.132 0.134 3.385 0.032
572 74511 SENSE 0.265 0.011 0.667 0.423
563 74063 SENSE 0.244 0.005 4.000 0.000
564 74072 SENSE 0.245 0.005 2.536 0.225
565 74073 SENSE 0.352 0.065 3.019 0.091
566 74075 SENSE 0.240 0.026 4.000 0.000
478 74445 SENSE 0.223 0.073 2.618 0.064
571 74490 SENSE 0.143 0.016 4.000 0.000
579 74703 SENSE 0.468 0.024 3.125 0.070
580 74733 SENSE 0.487 0.020 2.504 0.088
581 74749 SENSE 0.501 0.026 3.406 0.029
582 74752 SENSE 0.276 0.097 3.389 0.031
484 74755 SENSE 0.136 0.392 2.821 0.045
583 74773 SENSE 0.376 0.004 4.000 0.000
795 75867 SENSE 0.401 0.061 4.000 0.000
793 75530 SENSE −0.032 0.760 2.385 0.098
588 76003 SENSE 0.239 0.021 1.611 0.315
589 76074 SENSE 0.269 0.033 2.696 0.174
590 76137 SENSE 0.316 0.075 2.624 0.081
591 76220 SENSE 0.074 0.516 2.585 0.089
796 75910 SENSE 0.225 0.070 4.000 0.000
592 76301 SENSE 0.110 0.308 2.683 0.055
593 76318 SENSE 0.052 0.600 2.692 0.078
594 76347 SENSE 0.088 0.362 3.254 0.049
596 76566 SENSE 0.144 0.052 2.408 0.117
595 76513 SENSE 0.316 0.019 3.347 0.036
597 76719 SENSE 0.099 0.063 4.000
598 76757 SENSE 0.251 0.037 3.042 0.086
514 76917 SENSE 0.165 0.055 1.834 0.254
599 76891 SENSE 0.382 0.070 0.000 1.000
600 77208 SENSE 0.408 0.034 2.835 0.051

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
H. Shade Tolerance Screen
Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response may be associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½× MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m2/s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1 L.
Table 10 provides a list of recombinant DNA constructs that improve shade tolerance in plants
TABLE 10
Leaf Seedling Petiole
angle weight length
PEP at day at day at day
SEQ 23 23 23
ID Construct RS Delta Delta
NO ID Orientation mean P-value mean P-value mean P-value
441 70605 SENSE / / 0.424 0.061 0.352 0.038
632 72414 SENSE 0.000 1.000 0.396 0.067 0.222 0.047
464 73344 SENSE 2.667 0.057 0.179 0.444 0.289 0.058
744 73666 SENSE / / 0.838 0.008 0.142 0.445
453 72026 SENSE 0.000 1.000 0.469 0.042 0.313 0.073
634 73723 SENSE 1.333 0.423 −0.103 0.677 −0.406 0.089
635 73747 SENSE 2.000 0.225 −0.605 0.002 −0.565 0.050
633 73409 SENSE 2.667 0.057 0.365 0.052 0.283 0.010
636 74634 SENSE 0.667 0.423 −0.636 0.109 −0.869 0.017
615 74558 SENSE 1.333 0.184 0.486 0.018 0.231 0.108
824 74956 SENSE 1.333 0.423 0.154 0.076 −0.052 0.289
486 75220 SENSE 0.000 1.000 −0.644 0.230 −0.716 0.099
487 75226 SENSE 0.000 1.000 −0.601 0.067 −1.142 0.086
488 75231 SENSE 0.667 0.423 0.455 0.039 0.258 0.072
638 75232 SENSE 1.333 0.423 −1.931 0.077 −1.992 0.038
489 75239 SENSE 0.667 0.423 0.149 0.075 0.060 0.444
560 73855 SENSE 3.333 0.038 −0.261 0.121 −0.361 0.215
472 74059 SENSE 0.000 1.000 0.409 0.066 0.331 0.062
482 74707 SENSE 3.333 0.038 0.269 0.071 0.136 0.016
754 74753 SENSE 0.000 1.000 0.365 0.079 0.267 0.057
637 74769 SENSE 1.333 0.184 −0.141 0.346 −0.221 0.029
493 75355 SENSE 1.333 0.423 −0.735 0.069 −0.581 0.058
639 75429 SENSE 2.667 0.057 −0.453 0.151 −1.319 0.032
640 75965 SENSE 0.667 0.423 0.092 0.086 0.109 0.122
642 76061 SENSE / / 0.373 0.075 0.110 0.330
643 76155 SENSE 0.667 0.423 −0.473 0.021 −0.608 0.004
761 76207 SENSE 2.667 0.057 −0.374 0.060 −0.369 0.088
641 75975 SENSE 2.000 0.225 0.405 0.056 0.223 0.044
762 76293 SENSE 2.667 0.184 −0.412 0.052 −0.717 0.018
644 76322 SENSE 0.667 0.423 −0.533 0.044 −0.559 0.045
645 76440 SENSE / / −0.405 0.141 −0.525 0.045
509 76741 SENSE 0.667 0.423 −0.793 0.027 −0.803 0.018
513 76913 SENSE / / 0.313 0.073 0.336 0.129
646 76828 SENSE 0.667 0.423 −0.185 0.141 −1.189 0.001
517 77022 SENSE 0.000 1.000 −0.495 0.282 −0.475 0.079
648 77303 SENSE 2.000 0.225 −0.296 0.117 −0.176 0.038
649 77352 SENSE 1.333 0.423 −0.298 0.127 −0.377 0.036
631 77366 SENSE 0.667 0.423 0.611 0.015 0.327 0.044
647 77136 SENSE 1.333 0.423 −0.824 0.076 −0.566 0.089

For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2.
For “petiole length”, if p<0.05 and delta <0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta <0, the transgenic plants showed a trend of trait improvement as compared to the reference.
I. Early Plant Growth and Development Screen
This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 uE/m2/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
Table 11 provides a list recombinant DNA constructs that improve early plant growth and development.
TABLE 11
Root Root Seedling
PEP length at length at weight at
SEQ day 10 day 14 day 14
ID Construct Delta Delta Delta
NO ID Orientation mean P-value mean P-value mean P-value
523 14810 SENSE 0.175 0.027 0.049 0.231 −0.089 0.337
773 14836 SENSE 0.105 0.007 0.089 0.029 0.162 0.217
735 14944 SENSE 0.205 0.207 0.175 0.046 0.259 0.221
525 15419 ANTI-SENSE 0.182 0.019 0.125 0.048 0.212 0.155
527 17903 SENSE 0.401 0.008 0.174 0.011 0.330 0.005
529 18284 SENSE 0.031 0.795 0.016 0.722 0.223 0.043
737 18536 SENSE 0.158 0.222 0.104 0.118 0.149 0.122
736 18410 SENSE 0.222 0.228 0.078 0.640 0.141 0.080
432 17410 SENSE 0.145 0.052 0.074 0.246 0.074 0.542
532 18623 SENSE 0.247 0.100 0.133 0.074 0.228 0.036
434 18665 SENSE 0.237 0.025 0.156 0.057 0.268 0.033
738 19540 SENSE 0.115 0.397 0.085 0.237 0.130 0.031
739 19624 SENSE 0.329 0.057 0.171 0.269 0.494 0.024
740 70353 SENSE 0.136 0.065 0.086 0.355 0.140 0.170
780 70804 SENSE 0.411 0.066 0.290 0.147 0.483 0.129
443 70812 SENSE 0.438 0.069 0.351 0.043 0.571 0.036
444 70933 SENSE 0.093 0.099 0.088 0.039 0.071 0.060
540 70943 SENSE 0.344 0.074 0.173 0.031 0.263 0.283
741 71215 SENSE 0.282 0.107 0.142 0.010 0.108 0.594
446 71319 SENSE 0.201 0.077 0.051 0.427 0.236 0.025
742 71320 SENSE 0.601 0.015 0.437 0.041 0.547 0.107
447 71446 SENSE 0.308 0.182 0.207 0.021 0.465 0.107
463 73338 SENSE 0.309 0.035 0.146 0.038 0.462 0.017
611 73619 SENSE 0.070 0.151 0.103 0.016 0.266 0.093
744 73666 SENSE 0.304 0.036 0.166 0.014 0.480 0.010
469 73733 SENSE 0.156 0.148 −0.087 0.511 0.156 0.036
612 73737 SENSE 0.352 0.005 0.244 0.020 0.098 0.093
557 73750 SENSE 0.156 0.033 0.111 0.047 0.216 0.040
465 73536 SENSE 0.461 0.007 0.261 0.006 0.330 0.024
550 72993 SENSE 0.254 0.034 0.152 0.228 0.271 0.073
747 74211 SENSE 0.155 0.084 0.068 0.159 0.368 0.034
569 74315 SENSE 0.328 0.007 0.149 0.020 0.513 0.055
748 74510 SENSE 0.277 0.003 0.158 0.016 0.433 0.029
749 74527 SENSE 0.446 0.025 0.256 0.022 0.412 0.052
750 74539 SENSE 0.103 0.024 0.063 0.196 0.249 0.013
576 74550 SENSE 0.216 0.076 0.228 0.035 0.424 0.048
481 74576 SENSE 0.270 0.017 0.016 0.823 0.216 0.235
752 74577 SENSE 0.333 0.032 0.224 0.023 0.285 0.094
743 73076 SENSE 0.129 0.459 0.063 0.513 0.244 0.055
756 74908 SENSE 0.237 0.006 0.184 0.014 0.253 0.094
751 74556 SENSE 0.147 0.111 0.076 0.025 0.377 0.035
489 75239 SENSE 0.180 0.145 0.085 0.019 0.236 0.162
758 75254 SENSE 0.144 0.233 0.031 0.502 0.363 0.054
553 73224 SENSE 0.384 0.035 0.257 0.022 0.616 0.029
745 73852 SENSE 0.029 0.760 0.031 0.617 0.031 0.019
572 74511 SENSE 0.345 0.003 0.107 0.069 0.487 0.043
565 74073 SENSE −0.003 0.969 −0.023 0.646 0.225 0.096
746 74080 SENSE 0.252 0.031 0.102 0.152 0.296 0.074
753 74729 SENSE 0.065 0.082 −0.031 0.642 0.080 0.068
581 74749 SENSE 0.160 0.161 0.104 0.120 0.243 0.322
754 74753 SENSE 0.136 0.127 0.061 0.224 0.422 0.002
755 74754 SENSE 0.216 0.004 0.133 0.040 0.188 0.055
484 74755 SENSE 0.397 0.071 0.243 0.084 0.499 0.037
757 75107 SENSE 0.352 0.014 0.212 0.044 0.342 0.086
759 75741 SENSE 0.160 0.098 0.062 0.399 0.123 0.320
760 75931 SENSE 0.162 0.037 0.134 0.012 0.225 0.061
797 76101 SENSE 0.142 0.445 0.109 0.361 0.372 0.051
761 76207 SENSE 0.397 0.015 0.235 0.100 0.151 0.149
762 76293 SENSE 0.160 0.172 0.178 0.042 0.491 0.000
495 75847 SENSE 0.262 0.003 0.005 0.928 0.246 0.159
763 76517 SENSE 0.220 0.080 0.162 0.021 0.210 0.017
764 76559 SENSE 0.283 0.130 0.211 0.123 0.266 0.093
765 76804 SENSE 0.219 0.061 0.130 0.066 0.419 0.003
766 77018 SENSE 0.338 0.002 0.137 0.095 0.469 0.059
522 77351 SENSE 0.052 0.566 0.060 0.290 0.343 0.091
518 77108 SENSE 0.000 0.998 0.095 0.080 0.067 0.085
767 77115 SENSE 0.339 0.102 0.173 0.044 0.788 0.056
768 77128 SENSE 0.360 0.075 0.174 0.170 0.304 0.125
769 77158 SENSE 0.290 0.052 0.033 0.484 0.365 0.010
770 77159 SENSE 0.303 0.019 0.177 0.166 0.408 0.033
771 77163 SENSE 0.497 0.007 0.434 0.002 1.047 0.038
600 77208 SENSE 0.206 0.011 0.103 0.077 0.388 0.009

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
J. Late Plant Growth and Development Screen
This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft3. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m2/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.
The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
Table 12 provides a list of recombinant DNA constructs that improve late plant growth and development.
TABLE 12
Rosette Seed Silique
dry Rosette net dry dry Silique
PEP weight at radius weight at weight at length
SEQ day 53 at day 25 day 62 day 53 at day 40
ID Delta P- Delta P- Delta P- Delta P- Delta P-
NO Orientation mean value mean value mean value mean value mean value
772 ANTI- 0.072 0.189 / / 0.846 0.002 0.143 0.381 −0.012 0.803
SENSE
773 SENSE 0.094 0.592 −0.238 0.098 0.665 0.059 0.247 0.474 −0.029 0.184
775 SENSE 0.134 0.019 / / 0.675 0.061 0.540 0.010 0.093 0.008
774 SENSE −0.262 0.112 −0.407 0.032 −0.393 0.120 0.357 0.081 0.003 0.922
776 SENSE −1.027 0.044 / / 2.216 0.004 0.351 0.355 0.135 0.001
780 SENSE 0.310 0.048 / / −0.245 0.629 0.149 0.157 0.149 0.043
781 SENSE 0.219 0.069 / / 0.779 0.001 −0.174 0.108 −0.015 0.595
777 SENSE 0.526 0.045 / / −0.388 0.478 0.131 0.281 −0.351 0.036
778 SENSE 0.402 0.004 −0.158 0.126 −2.159 0.036 −0.410 0.016 −0.195 0.043
779 SENSE 0.454 0.006 −0.124 0.234 −0.112 0.328 0.228 0.070 0.065 0.024
782 SENSE 0.195 0.205 −0.153 0.019 0.352 0.005 0.488 0.031 −0.063 0.473
783 SENSE −0.388 0.381 / / 1.339 0.002 −1.147 0.126 0.601 0.000
786 SENSE −0.107 0.280   0.091 0.148 0.563 0.058 0.166 0.165 0.042 0.084
787 SENSE −0.290 0.138 −0.752 0.020 0.501 0.044 −0.114 0.237 0.048 0.188
788 SENSE −0.064 0.709 −0.060 0.302 0.346 0.027 0.198 0.082 0.062 0.115
789 SENSE −0.060 0.585 −0.204 0.076 0.930 0.004 −0.243 0.109 0.094 0.016
790 SENSE −0.299 0.076   0.208 0.181 0.683 0.008 0.140 0.652 −0.047 0.070
792 SENSE 0.470 0.039 / / 0.113 0.691 −0.118 0.511 −0.003 0.921
791 SENSE −0.037 0.754 / / 0.484 0.084 −0.163 0.366 0.024 0.328
784 SENSE −0.182 0.070 −0.079 0.243 0.649 0.075 0.070 0.659 −0.150 0.112
785 SENSE −0.057 0.216 −0.086 0.016 1.645 0.008 0.278 0.036 0.015 0.364
795 SENSE 0.620 0.013 / / 0.698 0.010 −0.102 0.794 0.040 0.247
793 SENSE 0.492 0.045 −0.232 0.029 −0.505 0.030 0.025 0.881
794 SENSE −0.051 0.809 / / 0.809 0.003 −0.054 0.649 −0.020 0.786
797 SENSE 0.121 0.107 / / 0.915 0.017 −0.287 0.054 0.054 0.120
796 SENSE 0.324 0.017 / / −0.289 0.468 0.331 0.012 0.040 0.476
798 SENSE 0.411 0.038 / / 0.809 0.018 0.231 0.286 −0.048 0.279
799 SENSE −0.014 0.720 / / 0.337 0.064 −0.112 0.165 0.018 0.581

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
K. Limited Nitrogen Tolerance Screen
Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
T2 seeds were plated on glufosinate selection plates containing 0.5× N-Free Hoagland's T 0.1 mM NH4NO3 T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i. e., viable or non-viable) and root length. After 21 days of growth, plants were scored for BASTA resistance, visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1 L. The leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system. A statistical logistic regression model was developed to predict an overall value based on five colors for each plant.
Table 13 provides a list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants.
TABLE 13
PEP Root Leaf color Rosette
SEQ length Risk Weight
ID Construct Delta P- score P- Delta
NO ID Orientation mean value mean value mean P-value
650 10913 ANTI-SENSE −0.322 0.072 0.559 0.234 −0.052 0.209
651 10925 ANTI-SENSE −0.407 0.047 5.828 0.342 −0.064 0.416
652 11357 SENSE −0.436 0.016 0.996 0.047 −0.093 0.233
653 11358 SENSE −0.521 0.000 1.297 0.012 −0.003 0.945
654 11432 ANTI-SENSE −0.215 0.049 0.669 0.046 0.009 0.899
655 11927 ANTI-SENSE −0.416 0.028 0.581 0.021 0.028 0.293
430 11937 ANTI-SENSE −0.302 0.076 0.564 0.039 −0.102 0.067
656 12050 SENSE −0.188 0.003 1.323 0.032 −0.031 0.245
657 12358 ANTI-SENSE −0.179 0.050 0.278 0.065 0.024 0.818
658 13809 SENSE −0.126 0.024 0.796 0.303 −0.099 0.073
603 14733 SENSE −0.161 0.131 2.267 0.085 −0.055 0.676
525 15419 ANTI-SENSE −0.068 0.119 1.607 0.051 0.044 0.451
659 16877 SENSE −0.207 0.158 2.516 0.057 0.012 0.843
660 18117 ANTI-SENSE −0.221 0.004 0.906 0.004 −0.146 0.067
805 70819 SENSE −0.258 0.057 2.275 0.088 −0.103 0.231
777 70639 SENSE −0.276 0.071 −1.184 0.099 0.132 0.052
778 70668 SENSE −0.126 0.010 −0.521 0.156 0.067 0.520
661 71651 SENSE −0.037 0.599 0.485 0.042 −0.020 0.682
662 72449 SENSE −0.019 0.645 −0.738 0.043 0.324 0.024
454 72533 SENSE −0.011 0.866 1.131 0.017 0.068 0.237
663 72649 SENSE −0.154 0.309 0.181 0.093 −0.046 0.350
610 72978 SENSE −0.304 0.184 0.367 0.125 0.144 0.008
464 73344 SENSE −0.068 0.002 −0.547 0.400 0.040 0.612
744 73666 SENSE 0.239 0.137 −2.192 0.089 0.125 0.064
635 73747 SENSE −0.031 0.568 0.671 0.010 0.002 0.974
664 73107 SENSE −0.236 0.039 0.196 0.057 0.050 0.554
567 74135 SENSE 0.173 0.081 −2.988 0.061 0.117 0.090
636 74634 SENSE −1.049 0.019 0.805 0.083 −0.084 0.034
578 74644 SENSE −0.224 0.027 0.247 0.353 −0.054 0.256
665 74875 SENSE −0.297 0.201 1.084 0.089 0.201 0.106
666 75079 SENSE −0.303 0.277 0.583 0.071 −0.122 0.329
485 75202 SENSE −0.277 0.093 0.463 0.049 −0.020 0.570
668 75228 SENSE −0.838 0.036 1.239 0.033 −0.091 0.503
669 75246 SENSE −1.192 0.009 1.314 0.039 −0.136 0.205
809 73223 SENSE 0.060 0.764 −0.249 0.442 0.066 0.084
784 73283 SENSE 0.200 0.242 −2.819 0.046 0.198 0.051
637 74769 SENSE −0.135 0.090 0.642 0.024 0.102 0.064
667 75180 SENSE −0.266 0.031 0.602 0.021 0.006 0.281
670 75375 SENSE −0.647 0.012 1.618 0.001 −0.156 0.043
671 75476 SENSE −0.563 0.011 1.101 0.003 −0.091 0.162
673 75857 SENSE −0.256 0.067 2.472 0.006 −0.125 0.041
672 75502 SENSE 0.101 0.209 −0.147 0.482 0.156 0.066
675 75970 SENSE −0.125 0.191 0.963 0.036 0.046 0.391
676 76115 SENSE −0.499 0.113 0.619 0.092 0.018 0.752
833 76121 SENSE −0.707 0.012 1.695 0.007 −0.199 0.049
643 76155 SENSE −0.059 0.454 0.779 0.022 −0.033 0.742
677 76217 SENSE −0.312 0.120 0.750 0.001 −0.010 0.864
678 76287 SENSE −0.504 0.035 1.326 0.003 −0.147 0.018
679 76294 SENSE −0.492 0.002 1.096 0.006 −0.134 0.013
762 76293 SENSE 0.102 0.330 −2.184 0.008 0.358 0.049
674 75873 SENSE −0.191 0.071 3.070 0.093 0.109 0.197
680 76305 SENSE −0.643 0.001 2.319 0.000 −0.110 0.018
644 76322 SENSE −0.568 0.019 1.259 0.091 −0.205 0.019
837 76388 SENSE 0.078 0.155 −0.032 0.862 0.130 0.017
681 76391 SENSE −0.259 0.114 1.043 0.046 0.036 0.806
798 76431 SENSE 0.143 0.134 −0.383 0.554 0.196 0.034
682 76438 SENSE −0.183 0.029 0.790 0.057 −0.062 0.291
683 76452 SENSE −0.284 0.112 0.928 0.017 −0.002 0.969
684 76563 SENSE −0.032 0.100 −0.047 0.203 −0.095 0.153
839 76567 SENSE 0.228 0.057 −0.403 0.418 0.180 0.024
646 76828 SENSE −0.163 0.051 0.666 0.018 0.156 0.078
685 77055 SENSE −0.261 0.180 0.926 0.079 0.054 0.516
686 77059 SENSE −0.380 0.005 1.188 0.021 −0.138 0.012
687 77062 SENSE −0.494 0.020 1.284 0.033 0.010 0.759
688 77063 SENSE −0.290 0.018 0.814 0.019 −0.082 0.116
691 77314 SENSE −0.500 0.046 0.369 0.008 −0.185 0.029
692 77344 SENSE −0.163 0.029 0.199 0.016 0.067 0.350
693 77348 SENSE −0.218 0.003 0.313 0.000 0.002 0.960
846 77112 SENSE −0.025 0.839 −0.057 0.919 0.142 0.059
689 77218 SENSE −0.072 0.687 0.679 0.008 0.054 0.336
521 77219 SENSE 0.183 0.071 0.134 0.290 0.319 0.038
690 77256 SENSE −0.339 0.015 0.216 0.205 −0.058 0.302

For leaf color and rosette weight, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
L. Statistic Analysis for Qualitative Responses
Table 14 provides a list of responses that were analyzed as qualitative responses
TABLE 14
response screen categories (success vs. failure)
wilting response Risk Soil drought tolerance screen non-wilted vs. wilted
Score
growth stage at day 14 heat stress tolerance screen 50% of plants reach stage 1.03 vs. not
growth stage at day 14 salt stress tolerance screen 50% of plants reach stage 1.03 vs. not
growth stage at day 14 PEG induced osmotic stress tolerance 50% of plants reach stage 1.03 vs. not
screen
growth stage at day 7 cold germination tolerance screen 50% of plants reach stage 0.5 vs. not
number of rosette leaves Shade tolerance screen 5 leaves appeared vs. not
at day 23
flower bud formation at Shade tolerance screen flower buds appear vs. not
day 23
leaf angle at day 23 Shade tolerance screen >60 degree vs. <60 degree
number of green leaves at limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not
day 21
number of rosette leaves limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not
at day 21
Flower bud formation at limited nitrogen tolerance screen flower buds appear vs. not
day 21
Plants were grouped into transgenic and reference groups and were scored as success or failure according to Table 14. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log2 RR. Subsequently the risk scores from multiple events for each transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
M. Statistic Analysis for Quantitative Responses
Table 15 provides a list of responses that were analyzed as quantitative responses.
TABLE 15
response screen
seed yield Soil drought stress tolerance screen
seedling weight at day 14 heat stress tolerance screen
root length at day 14 heat stress tolerance screen
seedling weight at day 14 salt stress tolerance screen
root length at day 14 salt stress tolerance screen
root length at day 11 salt stress tolerance screen
seedling weight at day 14 PEG induced osmotic stress tolerance screen
root length at day 11 PEG induced osmotic stress tolerance screen
root length at day 14 PEG induced osmotic stress tolerance screen
rosette area at day 8 cold shock tolerance screen
rosette area at day 28 cold shock tolerance screen
difference in rosette area cold shock tolerance screen
from day 8 to day 28
root length at day 28 cold germination tolerance screen
seedling weight at day 23 Shade tolerance screen
petiole length at day 23 Shade tolerance screen
root length at day 14 Early plant growth and development screen
Seedling weight at day 14 Early plant growth and development screen
Rosette dry weight Late plant growth and development screen
at day 53
rosette radius at day 25 Late plant growth and development screen
seed dry weight at day 58 Late plant growth and development screen
silique dry weight at day 53 Late plant growth and development screen
silique length at day 40 Late plant growth and development screen
Seedling weight at day 21 Limited nitrogen tolerance screen
Root length at day 21 Limited nitrogen tolerance screen
The measurements (M) of each plant were transformed by log2 calculation. The Delta was calculated as log2M(transgenic)−log2M(reference). Subsequently the mean delta from multiple events of the transgene of interest was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. The Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
Example 2 Identification of Homologs
A BLAST searchable “All Protein Database” is constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” is constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database is queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 426 through SEQ ID NO: 850 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list is kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database is queried using amino acid sequences of SEQ ID NO: 426 through SEQ ID NO: 850 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value is compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 851 to SEQ ID NO: 33634. These orthologs are reported in Tables 2 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.
Example 3 Consensus Sequence Build
ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 426 and its homologs, through SEQ ID NO: 850 and its homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. The consensus sequence of SEQ ID NO: 601 and its 13 homologs was derived according to the procedure described above and is displayed in FIG. 1.
Example 4
This example illustrates the identification of amino acid domain by Pfam analysis.
The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO: 425 through 850 are shown in Table 16. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 488 is characterized by two Pfam domains, i.e. “NAF” and “Pyr_redox—2”. See also the protein with amino acids of SEQ ID NO: 441 which is characterized by three copies of the Pfam domain “zf-CCCH”. In Table 16 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 17.
TABLE 16
PEP
SEQ ID
NO GENE ID Pfam domain name begin stop score E-value
426 CGPG699 Histone 27 100 99.6 8.80E−27
427 CGPG567 MIF 2 115 69.4 1.00E−17
428 CGPG267 WD40 46 83 28.3 2.40E−05
428 CGPG267 WD40 136 173 36.3 9.60E−08
430 CGPG959 NPH3 209 444 429.9  3.20E−126
431 CGPG2158 LSM 14 92 75.7 1.30E−19
432 CGPG2446 NUDIX 63 210 94.9 2.20E−25
433 CGPG1862 Bromodomain 421 510 97.4 4.00E−26
435 CGPG1674 efhand 303 331 23.1 0.00089
435 CGPG1674 Na_Ca_ex 441 575 58.5 1.90E−14
436 CGPG2680 Linker_histone 23 93 101 3.30E−27
437 CGPG3577 Glyoxalase 13 132 45.2 2.00E−10
438 CGPG4065 LRR_2 150 174 17.3 0.051
439 CGPG3929 Lung_7-TM_R 134 419 130.2 5.20E−36
441 CGPG3012 zf-CCCH 30 57 7.7 0.12
441 CGPG3012 zf-CCCH 61 85 7.3 0.13
441 CGPG3012 zf-CCCH 115 140 19.3 0.0034
442 CGPG3162 SBF 128 313 239.7 5.60E−69
443 CGPG607 PurA 28 275 44.4 5.80E−12
444 CGPG4084 PCI 251 355 102 1.60E−27
445 CGPG3917 Pkinase 13 268 341.6 1.20E−99
445 CGPG3917 NAF 307 367 124.7 2.30E−34
446 CGPG4414 p450 32 502 364.5  1.50E−106
447 CGPG185 Cyclin_N 62 187 117.7 3.00E−32
447 CGPG185 Cyclin_C 189 312 83.3 7.00E−22
448 CGPG1679 Ferric_reduct 183 304 126.7 6.00E−35
448 CGPG1679 FAD_binding_8 334 435 163.6 4.70E−46
448 CGPG1679 FAD_binding_6 336 435 −7.5 0.0026
448 CGPG1679 NAD_binding_6 441 709 348.5  9.90E−102
449 CGPG271 PP2C 269 629 56 1.10E−13
450 CGPG4434 p450 30 490 347.1  2.70E−101
452 CGPG5253 SBP56 20 487 1316.4 0
453 CGPG5231 Pkinase 85 343 344.4  1.70E−100
453 CGPG5231 efhand 390 418 35.6 1.50E−07
453 CGPG5231 efhand 426 454 31.4 3.00E−06
453 CGPG5231 efhand 462 490 28.3 2.50E−05
453 CGPG5231 efhand 496 524 39.3 1.20E−08
455 CGPG4859 PSI_PsaF 47 221 443.9  2.00E−130
456 CGPG1589 PLAC8 292 390 87.4 4.00E−23
458 CGPG3899 Pkinase 132 417 294.1 2.40E−85
460 CGPG5665 Aminotran_3 27 362 550.2  2.00E−162
460 CGPG5665 Aminotran_1_2 42 417 −49.4 5.40E−05
461 CGPG5697 Aminotran_3 25 360 503.2  2.70E−148
461 CGPG5697 Aminotran_1_2 40 415 −51.6 7.00E−05
462 CGPG5695 Pyr_redox 148 240 10.4 0.0019
462 CGPG5695 Pyr_redox_2 148 450 71.6 2.20E−18
463 CGPG4862 Glyco_hydro_1 40 520 611  9.40E−181
465 CGPG6541 PGK 88 479 732.8  2.10E−217
466 CGPG1756 NOP5NT 2 67 119.5 8.70E−33
466 CGPG1756 NOSIC 160 212 127.9 2.70E−35
466 CGPG1756 Nop 252 400 332.4 7.00E−97
467 CGPG4927 WD40 294 331 32.9 1.00E−06
467 CGPG4927 WD40 336 373 24.7 0.0003
467 CGPG4927 WD40 378 428 22.1 0.0018
468 CGPG5106 C2 17 96 91.7 2.10E−24
469 CGPG5167 p450 43 501 265.6 9.30E−77
470 CGPG1924 F-box 21 68 39.6 9.80E−09
471 CGPG5201 Methyltransf_11 116 231 86.8 6.20E−23
471 CGPG5201 Methyltransf_12 116 229 31.3 3.10E−06
472 CGPG1884 Pkinase_Tyr 88 365 137.7 2.90E−38
472 CGPG1884 Pkinase 88 368 142.5 1.00E−39
473 CGPG5089 LRR_1 142 164 17.7 0.037
473 CGPG5089 LRR_1 166 188 8 8.2
473 CGPG5089 LRR_1 190 212 9.3 4.7
473 CGPG5089 LRR_1 214 233 10.7 2.6
473 CGPG5089 LRR_1 238 257 9.4 4.5
473 CGPG5089 Pkinase 402 658 −21.1 7.60E−07
474 CGPG5870 Pkinase 47 314 77.2 4.60E−20
474 CGPG5870 Pkinase_Tyr 48 314 67.5 8.20E−20
475 CGPG5888 Pkinase 12 270 119.6 7.90E−33
476 CGPG1461 PGAM 79 265 151.4 2.10E−42
477 CGPG6743 PGK 88 483 573.5  1.80E−169
478 CGPG6722 Gln-synt_C 208 468 370.2  2.90E−108
479 CGPG82 Pkinase 43 329 324.8 1.40E−94
480 CGPG6761 Pyr_redox 155 247 23.4 0.00017
480 CGPG6761 Pyr_redox_2 155 468 91.9 1.80E−24
481 CGPG6781 Aminotran_3 28 350 619.8  2.20E−183
482 CGPG4914 WD40 281 318 31 3.80E−06
483 CGPG5840 Pkinase 5 274 146.1 8.30E−41
484 CGPG5980 Pkinase 9 278 134.2 3.20E−37
485 CGPG1743 mTERF 88 391 536.8  2.00E−158
486 CGPG5434 MtN3_slv 9 98 76.7 6.50E−20
486 CGPG5434 MtN3_slv 132 206 56.5 8.10E−14
487 CGPG5824 Pkinase 121 407 304.3 2.00E−88
488 CGPG5879 Pkinase 74 328 349  7.00E−102
488 CGPG5879 NAF 383 441 95.5 1.50E−25
489 CGPG5949 Pyr_redox_2 6 302 154.8 2.00E−43
489 CGPG5949 Pyr_redox 164 259 97 5.30E−26
490 CGPG6096 GSHPx 79 187 229.1 8.70E−66
491 CGPG6218 MFS_1 27 449 95.1 2.00E−25
491 CGPG6218 Sugar_tr 30 488 528.1  8.40E−156
492 CGPG6226 Sugar_tr 101 556 315.4 9.20E−92
492 CGPG6226 MFS_1 105 515 81.2 2.90E−21
494 CGPG7654 HABP4_PAI-RBP1 159 272 149.9 6.00E−42
495 CGPG6875 zf-C3HC4 202 239 25.3 0.00019
496 CGPG8259 TIM 4 245 454.3  1.40E−133
498 CGPG8224 NIR_SIR_ferr 66 133 49.7 8.80E−12
498 CGPG8224 NIR_SIR 166 347 199.3 8.30E−57
498 CGPG8224 NIR_SIR_ferr 362 434 69.7 8.70E−18
498 CGPG8224 NIR_SIR 443 591 0.4 0.0014
499 CGPG1927 F-box 38 85 38.8 1.70E−08
499 CGPG1927 Arm 418 458 27 5.90E−05
499 CGPG1927 Arm 459 499 34.8 2.70E−07
499 CGPG1927 Arm 500 543 39.2 1.30E−08
499 CGPG1927 Arm 544 585 37.4 4.50E−08
499 CGPG1927 Arm 589 630 45.7 1.40E−10
499 CGPG1927 Arm 631 674 28.5 2.20E−05
499 CGPG1927 Arm 675 715 45.8 1.30E−10
499 CGPG1927 Arm 716 757 25 0.00025
500 CGPG5962 Glyco_hydro_14 95 523 269.8 5.00E−78
501 CGPG5375 IMPDH 18 493 696.2  2.10E−206
503 CGPG8943 MGS 123 238 189.9 5.50E−54
503 CGPG8943 AICARFT_IMPCHas 243 568 635.4  4.30E−188
504 CGPG8896 Ferric_reduct 122 279 184.5 2.40E−52
505 CGPG8960 DnaJ 77 139 86.9 5.60E−23
505 CGPG8960 Fer4 162 185 12.6 0.0035
506 CGPG5891 Pkinase 56 307 −19.6 6.30E−07
507 CGPG7260 ATP-grasp_2 5 204 −43.8 5.10E−08
508 CGPG2647 Fibrillarin 79 310 599.4  2.90E−177
509 CGPG6995 F-box 23 71 30.2 6.70E−06
510 CGPG9046 Pro_isomerase 36 195 57.4 4.40E−14
511 CGPG9047 Pec_lyase_C 108 274 124.5 2.80E−34
513 CGPG9076 Auxin_inducible 7 106 26.3 5.50E−08
515 CGPG9109 CS 19 93 91.2 2.80E−24
516 CGPG5933 Nodulin-like 16 263 497.5  1.40E−146
517 CGPG6335 Sterol_desat 35 246 238.8 1.10E−68
518 CGPG9174 YjeF_N 28 205 146.4 7.10E−41
518 CGPG9174 Carb_kinase 268 524 267.5 2.50E−77
519 CGPG9120 Glyoxal_oxid_N 114 355 530.2  2.10E−156
522 CGPG8087 DUF1677 43 159 228.8 1.10E−65
523 CGPG385 RRM_1 13 76 71.4 2.70E−18
523 CGPG385 zf-CCHC 99 116 34.1 4.40E−07
523 CGPG385 zf-CCHC 121 138 28 1.90E−05
524 CGPG1857 FHA 32 107 45.8 1.30E−10
525 CGPG1788 PB1 100 192 88.2 2.30E−23
526 CGPG1966 PHD 66 114 57.6 3.70E−14
526 CGPG1966 SET 238 373 99 1.30E−26
527 CGPG1908 F-box 12 59 31 3.80E−06
527 CGPG1908 LRR_2 171 197 24.5 0.00035
529 CGPG3557 DnaJ 4 67 139.6 7.80E−39
529 CGPG3557 DnaJ_C 214 336 53.7 5.70E−13
530 CGPG3340 Dehydrin 21 186 188.8 1.20E−53
531 CGPG3431 DUF914 3 330 829.1  2.20E−246
532 CGPG3530 DUF1644 29 181 359.3  5.80E−105
533 CGPG3119 DUF231 262 434 243.6 3.80E−70
534 CGPG3594 PRA-CH 81 155 109.1 1.20E−29
534 CGPG3594 PRA-PH 176 269 76 1.10E−19
535 CGPG4031 Pkinase_Tyr 73 355 134.8 2.20E−37
535 CGPG4031 Pkinase 73 355 173.8 4.10E−49
536 CGPG4036 Aa_trans 31 467 546.7  2.20E−161
537 CGPG2384 Epimerase 3 266 18.3 1.80E−07
537 CGPG2384 3Beta_HSD 4 292 −95.6 5.50E−06
538 CGPG1598 Hist_deacetyl 149 461 399.3  5.10E−117
539 CGPG560 zf-CCCH 43 69 33.8 5.50E−07
539 CGPG560 zf-CCCH 88 114 42 1.80E−09
539 CGPG560 zf-CCCH 134 160 43.1 8.60E−10
539 CGPG560 zf-CCCH 244 270 44.7 2.90E−10
539 CGPG560 zf-CCCH 290 316 46.2 9.80E−11
540 CGPG4002 Aldo_ket_red 15 319 258.8 1.00E−74
541 CGPG1422 ADH_N 33 148 131.6 1.90E−36
541 CGPG1422 ADH_zinc_N 179 314 106 9.80E−29
542 CGPG4429 p450 42 510 443  3.60E−130
544 CGPG2251 Abhydrolase_1 214 507 29.8 8.80E−06
545 CGPG646 2-Hacid_dh 31 357 64.1 4.10E−16
545 CGPG646 2-Hacid_dh_C 128 322 225.6 1.00E−64
546 CGPG2569 zf-CCCH 146 171 27.5 4.30E−05
546 CGPG2569 WD40 178 215 25.9 0.00013
546 CGPG2569 WD40 302 338 31.9 2.00E−06
546 CGPG2569 WD40 343 378 26.4 9.40E−05
547 CGPG5507 zf-MYND 74 111 47.9 3.10E−11
547 CGPG5507 UCH 539 844 192.1 1.20E−54
548 CGPG5547 Hrf1 66 313 533.2  2.60E−157
548 CGPG5547 Yip1 108 286 1.7 0.00014
549 CGPG5517 HEAT 178 214 14.2 0.44
549 CGPG5517 HEAT 497 533 17.8 0.036
550 CGPG5792 AA_permease 90 561 483.8  1.90E−142
551 CGPG5766 PHD 282 329 54.3 3.70E−13
552 CGPG5775 SEP 237 311 150.2 4.90E−42
552 CGPG5775 UBX 343 422 105.7 1.20E−28
554 CGPG4872 WD40 310 347 31.9 2.00E−06
555 CGPG6420 ADH_N 27 155 128.4 1.90E−35
555 CGPG6420 ADH_zinc_N 186 327 138.2 2.10E−38
556 CGPG6402 PALP 44 349 −9.2 2.50E−07
557 CGPG5073 MatE 49 209 124.5 2.70E−34
557 CGPG5073 MatE 270 433 104.4 3.20E−28
558 CGPG5091 Lectin_C 69 189 15.7 2.10E−05
558 CGPG5091 Pkinase 257 542 64.3 3.60E−16
558 CGPG5091 Pkinase_Tyr 283 542 69.1 6.30E−20
559 CGPG3570 TMEM14 4 107 204.8 1.90E−58
560 CGPG4342 Mito_carr 114 210 119.3 1.00E−32
560 CGPG4342 Mito_carr 214 301 100.5 4.60E−27
560 CGPG4342 Mito_carr 304 392 100.7 4.10E−27
563 CGPG4351 ADH_N 59 146 41.2 3.30E−09
563 CGPG4351 ADH_zinc_N 177 323 66.6 7.40E−17
564 CGPG4753 FKBP_C 43 137 190.9 2.80E−54
565 CGPG4757 DPBB_1 65 152 140.4 4.40E−39
565 CGPG4757 Pollen_allerg_1 163 240 133.5 5.20E−37
567 CGPG6624 YGGT 92 174 111.5 2.20E−30
568 CGPG3161 ABC_tran 92 280 155.8 1.00E−43
568 CGPG3161 ABC2_membrane 384 590 180.6 3.60E−51
569 CGPG3770 ARID 21 129 36.3 3.70E−08
569 CGPG3770 ELM2 372 427 33 9.50E−07
569 CGPG3770 Myb_DNA-binding 472 518 19.4 0.011
571 CGPG6702 Aldedh 103 564 794.5  5.60E−236
572 CGPG21 MIP 30 265 447.2  2.00E−131
573 CGPG6801 PEPCK_ATP 18 492 1215.7 0
574 CGPG154 Aa_trans 29 428 516.5  2.80E−152
575 CGPG6762 Aldedh 28 511 665.1  5.10E−197
576 CGPG6763 PK 1 343 804.6  5.00E−239
576 CGPG6763 PK_C 355 470 176.3 6.80E−50
576 CGPG6763 PEP-utilizers 486 574 116.7 6.00E−32
577 CGPG1467 Auxin_inducible 23 112 153 6.90E−43
578 CGPG6160 Miro 11 126 66.4 8.20E−17
578 CGPG6160 Ras 12 173 318 1.50E−92
579 CGPG15 p450 28 483 375.3  8.60E−110
580 CGPG5825 Pkinase 138 425 297.9 1.80E−86
581 CGPG5936 MFS_1 48 421 141.9 1.60E−39
582 CGPG5974 FMO-like 10 457 −195.5 5.40E−15
582 CGPG5974 DAO 12 288 −14.4 7.70E−05
582 CGPG5974 Pyr_redox_2 12 308 −16.4 0.0018
583 CGPG1366 Pkinase 87 363 126.5 6.90E−35
583 CGPG1366 Pkinase_Tyr 87 364 123.2 6.70E−34
584 CGPG7390 DapB_N 51 180 68.3 2.20E−17
584 CGPG7390 DapB_C 183 315 100.6 4.40E−27
585 CGPG7421 Ribul_P_3_epim 91 291 411.7  9.30E−121
585 CGPG7421 OMPdecase 94 300 −46 0.0036
586 CGPG7446 TPR_1 123 156 26.4 9.10E−05
586 CGPG7446 TPR_2 123 156 24.4 0.00038
586 CGPG7446 TPR_1 160 193 37.1 5.70E−08
586 CGPG7446 TPR_2 160 193 32.9 9.90E−07
586 CGPG7446 TPR_1 194 241 14.4 0.093
587 CGPG6295 Pkinase 67 345 −14.9 3.40E−07
588 CGPG1476 RRM_1 26 97 63.9 4.80E−16
588 CGPG1476 RRM_1 114 184 98.1 2.40E−26
588 CGPG1476 RRM_1 203 273 88.2 2.30E−23
588 CGPG1476 RRM_1 306 376 94.6 2.80E−25
588 CGPG1476 PABP 504 581 69.4 1.10E−17
589 CGPG1821 F-box 10 57 37.4 4.50E−08
589 CGPG1821 LRR_2 166 190 16.6 0.077
589 CGPG1821 LRR_2 376 401 6.6 1.9
590 CGPG6975 Trp_syntA 17 274 441.7  9.00E−130
591 CGPG6189 GDPD 43 321 186.4 6.40E−53
592 CGPG8868 Pkinase 49 318 114.4 2.90E−31
593 CGPG8909 Brix 29 346 270.9 2.30E−78
594 CGPG8951 Lipase_3 102 222 8.9 9.40E−05
595 CGPG5892 Pkinase 50 288 −29.2 2.20E−06
597 CGPG6142 FAD_binding_4 123 258 118.8 1.40E−32
599 CGPG9034 Nicastrin 249 458 357.9  1.50E−104
600 CGPG9270 Pkinase 241 513 133.9 4.00E−37
600 CGPG9270 Pkinase_Tyr 241 513 126.7 5.90E−35
602 CGPG1284 NAPRTase 172 439 196.4 6.20E−56
603 CGPG1640 DUF1639 117 190 80.8 3.80E−21
604 CGPG2136 HMA 13 73 52 1.80E−12
605 CGPG3542 Oxidored_FMN 11 346 315.7 7.60E−92
606 CGPG1691 Mlo 5 513 1236.3 0
607 CGPG4067 Glyco_transf_8 169 499 263 5.50E−76
608 CGPG5335 U-box 256 329 93.1 7.90E−25
608 CGPG5335 Arm 383 423 48.9 1.60E−11
608 CGPG5335 Arm 424 464 22.1 0.0018
608 CGPG5335 Arm 465 505 40.9 3.90E−09
608 CGPG5335 Arm 506 546 18.7 0.019
608 CGPG5335 Arm 547 587 34.1 4.30E−07
609 CGPG27 Ammonium_transp 43 467 685.5  3.50E−203
611 CGPG4375 Anti-silence 1 155 392.9  4.40E−115
612 CGPG5176 Pkinase 334 602 135.4 1.40E−37
612 CGPG5176 Pkinase_Tyr 334 606 124.9 2.00E−34
614 CGPG661 Flavodoxin_1 87 230 173.8 3.80E−49
614 CGPG661 FAD_binding_1 285 509 380.4  2.50E−111
614 CGPG661 NAD_binding_1 545 657 112 1.60E−30
615 CGPG869 ABC_tran 110 312 147.5 3.20E−41
616 CGPG6159 Miro 14 129 80.2 5.80E−21
616 CGPG6159 Ras 15 176 332.9 5.10E−97
618 CGPG6282 Pkinase_Tyr 86 365 140.5 4.10E−39
618 CGPG6282 Pkinase 86 365 167.8 2.60E−47
619 CGPG7671 GATase_2 2 259 109.8 7.20E−30
619 CGPG7671 SIS 356 490 123 7.60E−34
619 CGPG7671 SIS 527 666 83.5 6.10E−22
620 CGPG1574 SPX 1 293 370.2  2.90E−108
620 CGPG1574 EXS 550 718 320.5 2.80E−93
621 CGPG9294 Pkinase 22 281 169.6 7.20E−48
622 CGPG2435 Abhydrolase_3 92 307 250 4.50E−72
623 CGPG3336 GRP 1 109 144.7 2.30E−40
625 CGPG4168 zf-A20 10 34 29.1 1.40E−05
625 CGPG4168 zf-AN1 99 139 61 3.50E−15
626 CGPG6517 Aldedh 19 478 827.9  4.90E−246
627 CGPG993 p450 30 500 86.4 7.90E−23
629 CGPG5393 PALP 20 309 439.2  5.10E−129
630 CGPG1313 Di19 10 218 482.6  4.40E−142
631 CGPG8150 DnaJ 93 162 23.9 1.90E−05
631 CGPG8150 HSCB_C 176 250 102.1 1.50E−27
632 CGPG1661 FAE_3-kCoA_syn1 14 340 776.2  1.80E−230
633 CGPG6427 ADH_N 27 155 129.8 6.70E−36
633 CGPG6427 ADH_zinc_N 186 332 129.4 9.40E−36
634 CGPG4906 WD40 46 83 39.2 1.30E−08
635 CGPG5092 Lectin_legB 25 262 393.3  3.30E−115
635 CGPG5092 Pkinase 353 611 45.8 1.30E−10
635 CGPG5092 Pkinase_Tyr 353 611 65.9 1.10E−19
636 CGPG6146 DUF241 37 272 374.9  1.20E−109
637 CGPG6022 RRM_1 46 113 28.5 2.20E−05
637 CGPG6022 RRM_1 163 233 44.6 3.10E−10
638 CGPG5883 Pkinase 129 411 125.5 1.40E−34
638 CGPG5883 Pkinase_Tyr 129 411 86.8 6.00E−23
640 CGPG8248 NAD_binding_2 1 163 203 6.40E−58
640 CGPG8248 6PGD 167 301 −145.6 3.60E−09
641 CGPG8233 Sedlin_N 3 77 177.8 2.50E−50
645 CGPG6337 Sterol_desat 38 246 218.1 1.80E−62
646 CGPG9005 CH 14 115 54.8 2.60E−13
646 CGPG9005 EB1 200 247 76.6 7.30E−20
647 CGPG9208 CoA_trans 5 242 104.7 2.50E−28
648 CGPG4348 Xan_ur_permease 94 532 −11.6 1.00E−07
650 CGPG618 ADK 38 224 317.8 1.80E−92
650 CGPG618 ADK_lid 160 195 83.4 6.40E−22
651 CGPG251 p450 37 459 223.1 5.80E−64
652 CGPG636 Ion_trans_2 81 163 75.3 1.80E−19
652 CGPG636 Ion_trans_2 202 277 59.5 9.80E−15
654 CGPG287 B56 71 484 1074.3 0
655 CGPG893 DUF231 210 368 324.8 1.30E−94
657 CGPG657 PI-PLC-X 106 248 73.4 6.70E−19
657 CGPG657 C2 405 496 85.1 1.90E−22
658 CGPG1489 Enolase_N 3 139 232.7 7.20E−67
658 CGPG1489 Enolase_C 147 441 719.3  2.40E−213
660 CGPG1683 DUF568 89 227 302 9.90E−88
661 CGPG4685 MFS_1 40 446 143.1 6.60E−40
662 CGPG4729 Auxin_inducible 37 146 217.2 3.40E−62
663 CGPG4880 Trehalose_PPase 108 346 314.1 2.30E−91
664 CGPG5680 Aminotran_1_2 30 384 204.7 1.90E−58
665 CGPG7317 ADH_N 27 155 123 7.60E−34
665 CGPG7317 ADH_zinc_N 186 328 146.5 6.30E−41
666 CGPG4902 DUF260 15 116 240.1 4.30E−69
667 CGPG6720 Aldedh 116 581 794.9  4.30E−236
668 CGPG5850 Pkinase 75 349 170.9 2.80E−48
668 CGPG5850 Pkinase_Tyr 75 349 128.3 2.00E−35
669 CGPG6010 RRM_1 130 200 60 7.30E−15
669 CGPG6010 RRM_1 229 299 62.6 1.20E−15
670 CGPG7488 OPT 28 651 682.3  3.40E−202
672 CGPG7672 PTPA 86 387 526.8  2.10E−155
674 CGPG6988 Sulfotransfer_1 79 340 296.6 4.10E−86
675 CGPG1357 DUF26 77 131 85.6 1.40E−22
675 CGPG1357 DUF26 190 241 81.6 2.20E−21
675 CGPG1357 Pkinase_Tyr 326 598 144.7 2.30E−40
675 CGPG1357 Pkinase 326 598 158.3 1.80E−44
676 CGPG5918 ADH_N 35 150 102.7 1.00E−27
676 CGPG5918 ADH_zinc_N 181 315 81.3 2.80E−21
677 CGPG6020 La 107 166 135.1 1.70E−37
677 CGPG6020 RRM_1 195 278 39.7 9.50E−09
678 CGPG7032 Pkinase 18 302 169.8 6.20E−48
678 CGPG7032 Pkinase_Tyr 18 302 174.3 2.80E−49
679 CGPG7069 Na_Ca_ex 115 249 118.5 1.80E−32
679 CGPG7069 Na_Ca_ex 415 558 118.9 1.30E−32
680 CGPG8900 K_trans 1 302 64.8 2.30E−23
681 CGPG8923 Pkinase 134 418 303.1 4.80E−88
682 CGPG6296 Pkinase 113 376 169.5 7.50E−48
682 CGPG6296 Pkinase_Tyr 113 376 150.7 3.50E−42
685 CGPG6951 RNA_pol_Rpb8 9 145 314.2 2.10E−91
686 CGPG6994 Ion_trans_2 155 237 56.5 8.00E−14
686 CGPG6994 Ion_trans_2 279 354 44 4.80E−10
687 CGPG7019 Aldo_ket_red 10 325 −73.8 5.00E−05
689 CGPG9255 Pkinase 97 417 246.9 3.90E−71
690 CGPG9274 CS 73 150 66.2 9.80E−17
690 CGPG9274 SGS 154 212 58.1 2.70E−14
691 CGPG6130 Miro 107 222 145.6 1.20E−40
691 CGPG6130 Ras 108 274 −24.6 1.80E−07
692 CGPG8074 PMEI 32 193 190.9 2.70E−54
694 CGPG172 WD40 187 222 23.4 0.00074
696 CGPG3222 zf-C3HC4 110 151 30.9 4.10E−06
697 CGPG3259 Asp 86 507 507.1  1.90E−149
697 CGPG3259 SapB_2 319 353 56.5 8.10E−14
697 CGPG3259 SapB_1 379 417 57 5.70E−14
698 CGPG1900 F-box 1 48 39.5 1.00E−08
700 CGPG20 MIP 30 259 448.1  1.10E−131
701 CGPG201 PI-PLC-X 113 257 137 4.80E−38
701 CGPG201 PI-PLC-Y 299 417 97.2 4.40E−26
701 CGPG201 C2 439 531 80.2 5.70E−21
702 CGPG3420 WD40 573 612 26.9 6.50E−05
703 CGPG4308 NPH3 30 309 254 2.90E−73
704 CGPG5244 FH2 589 985 601.3  8.30E−178
705 CGPG5538 aPHC 13 313 625.3  4.60E−185
706 CGPG3738 Glycolytic 55 399 842.5  1.90E−250
707 CGPG6454 PGM_PMM_I 103 249 157.3 3.70E−44
707 CGPG6454 PGM_PMM_II 280 394 163.5 4.90E−46
707 CGPG6454 PGM_PMM_III 396 518 150.6 3.70E−42
707 CGPG6454 PGM_PMM_IV 539 647 96.5 7.50E−26
708 CGPG6530 Glutaminase 121 412 609.3  3.10E−180
709 CGPG5175 p450 36 467 55.8 1.30E−13
710 CGPG5756 Pkinase 504 838 165.3 1.40E−46
711 CGPG5361 U-box 32 106 87.5 3.80E−23
712 CGPG6709 Aldedh 103 562 699.5  2.10E−207
713 CGPG6755 Aminotran_3 123 449 313.4 3.80E−91
714 CGPG6765 Alpha-amylase 16 418 541.6  7.40E−160
715 CGPG6039 LEA_2 2 151 358.6  8.90E−105
716 CGPG6158 Miro 9 124 78.5 1.90E−20
716 CGPG6158 Ras 10 171 357.2  2.40E−104
718 CGPG7528 Agenet 3 68 34 4.80E−07
718 CGPG7528 Agenet 141 206 93.6 5.50E−25
719 CGPG7756 BRAP2 51 161 137.8 2.70E−38
719 CGPG7756 zf-C3HC4 168 207 40.1 7.20E−09
719 CGPG7756 zf-UBP 219 290 119.4 9.30E−33
721 CGPG8249 PfkB 3 313 214 3.20E−61
722 CGPG2232 PCI 293 397 103.9 4.20E−28
723 CGPG5494 FAE1_CUT1_RppA 22 313 586  3.30E−173
723 CGPG5494 Chal_sti_synt_C 287 426 0 0.0016
723 CGPG5494 ACP_syn_III_C 333 424 31.3 7.50E−09
724 CGPG5947 Glyco_hydro_14 109 534 645.4  4.40E−191
726 CGPG6262 Pkinase 68 304 −46.3 2.00E−05
727 CGPG6909 2OG-FeII_Oxy 205 302 109.4 9.80E−30
729 CGPG8965 DnaJ 6 68 116.2 8.30E−32
730 CGPG5861 Pkinase 68 337 98.7 1.60E−26
730 CGPG5861 Pkinase_Tyr 68 337 78.1 1.40E−20
732 CGPG7246 MIP 75 285 170.5 3.80E−48
733 CGPG5378 WWE 77 148 88.4 2.00E−23
734 CGPG9168 NTP_transferase 5 286 481.1  1.20E−141
734 CGPG9168 MannoseP_isomer 297 462 449.5  4.10E−132
734 CGPG9168 Cupin_2 377 447 52 1.80E−12
735 CGPG1505 PGI 52 548 1050.2 0
737 CGPG3220 Methyltransf_6 2 159 181.8 1.60E−51
738 CGPG3062 Sugar_tr 26 489 672.8  2.30E−199
738 CGPG3062 MFS_1 32 450 89.6 8.70E−24
739 CGPG3580 DUF588 27 169 202.1 1.20E−57
740 CGPG3725 DUF1325 8 269 487.8  1.20E−143
741 CGPG592 Spermine_synth 50 295 493.4  2.40E−145
742 CGPG4417 p450 34 510 291.7 1.30E−84
743 CGPG2276 IF2_N 419 470 46.9 6.20E−11
743 CGPG2276 GTP_EFTU 499 670 157 4.40E−44
743 CGPG2276 Ras 513 668 −68.7 0.00036
743 CGPG2276 GTP_EFTU_D2 693 756 59 1.40E−14
744 CGPG4975 CK_II_beta 96 270 447.9  1.20E−131
745 CGPG2790 Asp 82 476 −116.5 9.10E−07
746 CGPG4972 PP2C 88 349 281 2.00E−81
747 CGPG5140 p450 27 502 87.5 3.70E−23
748 CGPG19 MIP 11 232 391.4  1.20E−114
749 CGPG6769 iPGM_N 12 367 828.1  4.30E−246
749 CGPG6769 Metalloenzyme 377 493 184.1 3.00E−52
750 CGPG6770 Molybdop_Fe4S4 53 114 70.4 5.30E−18
750 CGPG6770 Molybdopterin 117 670 82.9 8.90E−22
750 CGPG6770 Molydop_binding 902 1021 63.2 7.60E−16
752 CGPG6789 Isoamylase_N 12 98 134.2 3.20E−37
752 CGPG6789 Alpha-amylase 138 574 49.2 1.10E−12
753 CGPG5374 CBS 54 268 34 4.80E−07
753 CGPG5374 CBS 291 426 80.3 5.60E−21
754 CGPG5978 Pkinase_Tyr 74 349 229.9 5.00E−66
754 CGPG5978 Pkinase 74 349 185.3 1.30E−52
755 CGPG5979 Pkinase 108 369 219.6 6.40E−63
757 CGPG948 NPH3 165 407 469.9  2.90E−138
758 CGPG6052 DUF1723 61 110 92.8 9.50E−25
759 CGPG7661 HMA 91 154 50.4 5.40E−12
759 CGPG7661 DAO 188 525 −20.6 0.0002
759 CGPG7661 Pyr_redox_2 188 497 237.8 2.10E−68
759 CGPG7661 Pyr_redox 360 450 89 1.30E−23
759 CGPG7661 Pyr_redox_dim 525 634 160.9 3.00E−45
760 CGPG8261 Aminotran_3 27 350 484.5  1.20E−142
761 CGPG5447 Cys_Met_Meta_PP 176 561 752.1  3.20E−223
761 CGPG5447 Beta_elim_lyase 215 461 −106.2 0.0013
762 CGPG7068 DUF1005 249 432 467.9  1.10E−137
763 CGPG5938 MFS_1 45 420 78.2 2.30E−20
764 CGPG7100 BCNT 149 228 153.6 4.90E−43
765 CGPG9003 SNARE 37 99 66.1 1.00E−16
766 CGPG6299 Pkinase_Tyr 73 349 136 9.50E−38
766 CGPG6299 Pkinase 73 349 139.1 1.10E−38
767 CGPG9135 Acyl-CoA_dh_N 51 162 83.6 5.80E−22
767 CGPG9135 Acyl-CoA_dh_M 166 218 85.2 1.80E−22
767 CGPG9135 Acyl-CoA_dh_1 272 418 95.2 1.80E−25
767 CGPG9135 Acyl-CoA_dh_2 284 407 −9.9 0.0016
771 CGPG9139 TB2_DP1_HVA22 2 98 38.6 6.60E−09
772 CGPG414 Pkinase 80 338 353.1  4.00E−103
774 CGPG1986 Sulfotransfer_1 71 332 273.5 3.80E−79
775 CGPG2916 PC4 68 158 190.2 4.70E−54
776 CGPG111 PBP 20 165 256.9 3.80E−74
777 CGPG4317 zf-AN1 102 142 70.1 6.60E−18
778 CGPG4387 zf-A20 31 55 40.6 4.90E−09
778 CGPG4387 zf-AN1 124 164 64.7 2.80E−16
779 CGPG4492 Oleosin 27 145 87.2 4.70E−23
780 CGPG597 Glyco_hydro_28 98 437 482.9  3.60E−142
781 CGPG345 TPR_2 491 524 25 0.00024
781 CGPG345 TPR_1 491 524 18.8 0.018
781 CGPG345 TPR_2 590 623 25.4 0.00018
781 CGPG345 TPR_1 590 623 32.3 1.60E−06
781 CGPG345 TPR_1 624 657 24 0.00048
781 CGPG345 TPR_2 658 691 32.6 1.30E−06
781 CGPG345 TPR_1 658 691 36.6 8.00E−08
783 CGPG4998 LRRNT_2 27 67 57.2 5.00E−14
783 CGPG4998 LRR_1 71 93 9.9 3.6
783 CGPG4998 LRR_1 95 117 17.8 0.035
783 CGPG4998 LRR_1 119 141 17.2 0.056
783 CGPG4998 LRR_1 143 165 12.8 1
785 CGPG5104 C2 8 87 94.8 2.30E−25
786 CGPG6502 Pribosyltran 95 241 137.3 3.90E−38
787 CGPG6630 Lactamase_B 113 278 42.4 1.40E−09
788 CGPG5484 PP2C 100 340 253.9 3.10E−73
789 CGPG5394 PDT 101 278 281.4 1.60E−81
789 CGPG5394 ACT 288 371 26.6 8.30E−05
791 CGPG2090 DUF786 5 107 220.1 4.60E−63
792 CGPG6664 FeThRed_A 91 157 166.5 6.10E−47
793 CGPG7706 Acetyltransf_1 58 149 71.2 3.10E−18
794 CGPG7884 Aldedh 72 516 216.4 5.70E−62
795 CGPG6965 COX5C 2 63 162.4 1.10E−45
797 CGPG2297 TCTP 1 165 320.9 2.00E−93
798 CGPG6246 Pkinase 24 278 334.9 1.30E−97
798 CGPG6246 NAF 311 373 119.5 8.90E−33
799 CGPG9037 Ribophorin_I 40 472 658  6.90E−195
800 CGPG1941 TBC 271 503 −52.5 0.00069
801 CGPG2055 Terpene_synth 1 105 −37 7.70E−05
803 CGPG386 Ank 57 89 46.8 6.50E−11
803 CGPG386 Ank 91 123 22.6 0.0013
803 CGPG386 RCC1 259 307 29 1.50E−05
803 CGPG386 RCC1 311 361 25.7 0.00015
804 CGPG393 CDI 155 208 92.1 1.50E−24
805 CGPG609 Smr 428 502 81.8 2.00E−21
806 CGPG4022 Aa_trans 41 435 323.9 2.60E−94
807 CGPG942 AMP-binding 32 441 405  9.70E−119
808 CGPG1800 PHD 605 653 48.3 2.30E−11
810 CGPG3257 LRRNT_2 26 63 39.9 7.80E−09
810 CGPG3257 LRR_1 91 113 18.9 0.016
810 CGPG3257 LRR_1 115 137 12.2 1.4
810 CGPG3257 LRR_1 163 183 9.6 4.1
810 CGPG3257 Pkinase_Tyr 344 612 68.3 7.20E−20
810 CGPG3257 Pkinase 345 612 63.9 4.90E−16
811 CGPG1696 MtN3_slv 6 93 133.5 5.40E−37
811 CGPG1696 MtN3_slv 128 214 96.2 8.80E−26
813 CGPG3780 Response_reg 78 194 79.9 7.10E−21
813 CGPG3780 CCT 675 713 71.5 2.40E−18
814 CGPG6602 RuBisCO_large_N 104 229 293.3 4.30E−85
814 CGPG6602 RuBisCO_large 237 545 816.5  1.30E−242
815 CGPG6621 GIDA 128 453 −213 0.0004
815 CGPG6621 Pyr_redox_2 128 437 209.4 7.80E−60
815 CGPG6621 Pyr_redox 296 391 126.7 6.00E−35
815 CGPG6621 Pyr_redox_dim 470 579 158.4 1.70E−44
816 CGPG5493 FAE1_CUT1_RppA 113 402 727.7  7.00E−216
816 CGPG5493 Chal_sti_synt_C 384 528 −2.9 0.0028
816 CGPG5493 ACP_syn_III_C 442 526 23.2 5.20E−08
817 CGPG5811 Pkinase 107 381 198.8 1.20E−56
817 CGPG5811 Pkinase_Tyr 107 381 253.2 4.80E−73
818 CGPG5902 ADH_N 27 108 66.2 9.80E−17
818 CGPG5902 ADH_zinc_N 139 281 165.3 1.50E−46
819 CGPG6791 PGI 25 480 184.8 2.00E−52
820 CGPG6778 Alpha-amylase 13 420 442.8  4.00E−130
821 CGPG6166 Miro 7 121 52.9 1.00E−12
821 CGPG6166 Ras 8 177 266.5 4.90E−77
822 CGPG3735 LisH 47 73 43.8 5.40E−10
823 CGPG5854 Pkinase 2 275 97.2 4.60E−26
825 CGPG5024 adh_short 30 212 15.2 3.60E−07
826 CGPG6054 Miro 6 120 63.4 6.90E−16
826 CGPG6054 Ras 7 178 255.3 1.20E−73
827 CGPG6207 Sugar_tr 34 479 468.3  8.90E−138
827 CGPG6207 MFS_1 38 438 121.3 2.50E−33
828 CGPG7620 Arginase 69 349 375.5  7.30E−110
831 CGPG73 Dicty_CAR 10 314 −6.3 2.80E−06
832 CGPG2100 PLAC8 17 116 142.7 9.00E−40
833 CGPG6026 RRM_1 8 62 43.4 7.20E−10
834 CGPG7269 Tim17 18 146 146.8 5.20E−41
836 CGPG6993 Peptidase_C12 12 219 381.6  1.10E−111
838 CGPG6926 Yip1 93 240 161.1 2.70E−45
839 CGPG7172 Pkinase 3 269 192.5 9.30E−55
840 CGPG7129 DUF1070 9 63 115.5 1.40E−31
842 CGPG9031 MFAP1_C 141 423 511  1.20E−150
843 CGPG9105 ARD 14 168 329.7 4.50E−96
843 CGPG9105 Cupin_2 88 162 28.3 2.50E−05
844 CGPG9082 DUF662 7 169 236.3 6.00E−68
845 CGPG6212 Sugar_tr 20 490 52.4 1.40E−12
845 CGPG6212 MFS_1 29 451 98.9 1.40E−26
846 CGPG9206 Carboxyl_trans 34 535 1022.5  1.30E−304
847 CGPG9151 Aminotran_3 82 421 269 8.90E−78
848 CGPG9129 HATPase_c 228 356 83.7 5.20E−22
850 CGPG9358 TPR_1 551 584 10.5 0.28
850 CGPG9358 TPR_1 585 618 15.5 0.068
850 CGPG9358 TPR_1 655 688 16.1 0.059
TABLE 17
Pfam domain accession gathering
name number cutoff domain description
zf-MYND PF01753.8 11 MYND finger
UCH PF00443.18 −8.6 Ubiquitin carboxyl-terminal hydrolase
MIF PF01187.7 −17.6 Macrophage migration inhibitory factor (MIF)
PurA PF04845.3 25 PurA ssDNA and RNA-binding protein
Gln-synt_C PF00120.14 −124 Glutamine synthetase, catalytic domain
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
NAF PF03822.4 4.5 NAF domain
Sterol_desat PF01598.7 −13 Sterol desaturase
Pkinase PF00069.14 −70.8 Protein kinase domain
Asp PF00026.13 −186.1 Eukaryotic aspartyl protease
Glyco_hydro_1 PF00232.9 −301.8 Glycosyl hydrolase family 1
Oleosin PF01277.7 −27 Oleosin
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
ATP-grasp_2 PF08442.1 −118.8 ATP-grasp domain
PLAC8 PF04749.6 −1.1 PLAC8 family
Ferric_reduct PF01794.8 −7 Ferric reductase like transmembrane
component
FAD_binding_8 PF08022.1 −10.4 FAD-binding domain
FAD_binding_6 PF00970.13 −11.4 Oxidoreductase FAD-binding domain
NAD_binding_6 PF08030.1 −23.6 Ferric reductase NAD binding domain
LSM PF01423.12 13.7 LSM domain
Fibrillarin PF01269.7 −86.6 Fibrillarin
WD40 PF00400.20 21.5 WD domain, G-beta repeat
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Linker_histone PF00538.8 −8 linker histone H1 and H5 family
PP2C PF00481.11 −44 Protein phosphatase 2C
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
SBF PF01758.6 −27.8 Sodium Bile acid symporter family
Glyoxalase PF00903.14 12.1 Glyoxalase/Bleomycin resistance
protein/Dioxygenase superfamily
Pkinase PF00069.14 −70.8 Protein kinase domain
NAF PF03822.4 4.5 NAF domain
LRR_2 PF07723.2 6 Leucine Rich Repeat
p450 PF00067.11 −105 Cytochrome P450
WD40 PF00400.20 21.5 WD domain, G-beta repeat
WD40 PF00400.20 21.5 WD domain, G-beta repeat
WD40 PF00400.20 21.5 WD domain, G-beta repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
Pkinase PF00069.14 −70.8 Protein kinase domain
C2 PF00168.18 3.7 C2 domain
SBP56 PF05694.1 25 56 kDa selenium binding protein (SBP56)
IMPDH PF00478.13 −190.6 IMP dehydrogenase/GMP reductase domain
MtN3_slv PF03083.5 −0.8 MtN3/saliva family
MtN3_slv PF03083.5 −0.8 MtN3/saliva family
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
NAF PF03822.4 4.5 NAF domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Nodulin-like PF06813.3 −57.8 Nodulin-like
Glyco_hydro_14 PF01373.7 −231.4 Glycosyl hydrolase family 14
GSHPx PF00255.10 −16 Glutathione peroxidase
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
PGK PF00162.9 −39.9 Phosphoglycerate kinase
PGK PF00162.9 −39.9 Phosphoglycerate kinase
Pyr_redox PF00070.17 5 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)
MIP PF00230.9 −62 Major intrinsic protein
Tim17 PF02466.8 2.7 Tim17/Tim22/Tim23 family
HABP4_PAI-RBP1 PF04774.4 17.1 Hyaluronan/mRNA binding family
DUF1677 PF07911.4 25 Protein of unknown function (DUF1677)
NIR_SIR_ferr PF03460.6 2.4 Nitrite/Sulfite reductase ferredoxin-like half
domain
NIR_SIR PF01077.11 −25 Nitrite and sulphite reductase 4Fe—4S domain
NIR_SIR_ferr PF03460.6 2.4 Nitrite/Sulfite reductase ferredoxin-like half
domain
NIR_SIR PF01077.11 −25 Nitrite and sulphite reductase 4Fe—4S domain
TIM PF00121.8 −97 Triosephosphate isomerase
DnaJ PF00226.19 −8 DnaJ domain
Fer4 PF00037.15 9.3 4Fe—4S binding domain
Pro_isomerase PF00160.10 −37 Cyclophilin type peptidyl-prolyl cis-trans
isomerase/CLD
Pec_lyase_C PF00544.8 −45 Pectate lyase
CS PF04969.5 8.6 CS domain
Glyoxal_oxid_N PF07250.1 25 Glyoxal oxidase N-terminus
YjeF_N PF03853.4 25 YjeF-related protein N-terminus
Carb_kinase PF01256.7 −66.3 Carbohydrate kinase
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
DUF1644 PF07800.2 25 Protein of unknown function (DUF1644)
TMEM14 PF03647.3 −1 Transmembrane proteins 14C
Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family
Pkinase PF00069.14 −70.8 Protein kinase domain
BCNT PF07572.2 25 Bucentaur or craniofacial development
Mito_carr PF00153.15 0 Mitochondrial carrier protein
Mito_carr PF00153.15 0 Mitochondrial carrier protein
Mito_carr PF00153.15 0 Mitochondrial carrier protein
NUDIX PF00293.17 0 NUDIX domain
PRA-CH PF01502.9 25 Phosphoribosyl-AMP cyclohydrolase
PRA-PH PF01503.8 6 Phosphoribosyl-ATP pyrophosphohydrolase
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
WD40 PF00400.20 21.5 WD domain, G-beta repeat
WD40 PF00400.20 21.5 WD domain, G-beta repeat
WD40 PF00400.20 21.5 WD domain, G-beta repeat
CK_II_beta PF01214.9 −106 Casein kinase II regulatory subunit
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
Yip1 PF04893.6 −6.4 Yip1 domain
ARID PF01388.11 −8 ARID/BRIGHT DNA binding domain
ELM2 PF01448.12 12 ELM2 domain
Myb_DNA-binding PF00249.19 2.8 Myb-like DNA-binding domain
PP2C PF00481.11 −44 Protein phosphatase 2C
MatE PF01554.8 59.6 MatE
MatE PF01554.8 59.6 MatE
Methyltransf_11 PF08241.1 17.1 Methyltransferase domain
Methyltransf_12 PF08242.1 21.4 Methyltransferase domain
FMO-like PF00743.9 −381.6 Flavin-binding monooxygenase-like
DAO PF01266.12 −35.9 FAD dependent oxidoreductase
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
Auxin_inducible PF02519.4 −15 Auxin responsive protein
Response_reg PF00072.12 4 Response regulator receiver domain
CCT PF06203.4 25 CCT motif
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
PABP PF00658.8 25 Poly-adenylate binding protein, unique
domain
p450 PF00067.11 −105 Cytochrome P450
Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter
protein
F-box PF00646.21 13.6 F-box domain
LRR_2 PF07723.2 6 Leucine Rich Repeat
LRR_2 PF07723.2 6 Leucine Rich Repeat
FHA PF00498.14 25 FHA domain
Bromodomain PF00439.14 8.9 Bromodomain
PHD PF00628.17 25.9 PHD-finger
SET PF00856.17 23.5 SET domain
Abhydrolase_1 PF00561.10 10.3 alpha/beta hydrolase fold
Epimerase PF01370.11 −46.3 NAD dependent epimerase/dehydratase
family
3Beta_HSD PF01073.8 −135.9 3-beta hydroxysteroid
dehydrogenase/isomerase family
DUF231 PF03005.5 −58 Arabidopsis proteins of unknown function
ABC_tran PF00005.15 9.5 ABC transporter
ABC2_membrane PF01061.12 −17.9 ABC-2 type transporter
Dehydrin PF00257.9 −4.4 Dehydrin
DnaJ PF00226.19 −8 DnaJ domain
DnaJ_C PF01556.9 −24 DnaJ C terminal region
Ank PF00023.18 0 Ankyrin repeat
Ank PF00023.18 0 Ankyrin repeat
RCC1 PF00415.8 19 Regulator of chromosome condensation
(RCC1)
RCC1 PF00415.8 19 Regulator of chromosome condensation
(RCC1)
Pkinase PF00069.14 −70.8 Protein kinase domain
Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptor
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter
protein
PCI PF01399.15 25 PCI domain
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
p450 PF00067.11 −105 Cytochrome P450
FKBP_C PF00254.17 −7.6 FKBP-type peptidyl-prolyl cis-trans isomerase
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Lectin_C PF00059.10 −10.4 Lectin C-type domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
HEAT PF02985.10 9.9 HEAT repeat
HEAT PF02985.10 9.9 HEAT repeat
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
PHD PF00628.17 25.9 PHD-finger
SEP PF08059.2 25 SEP domain
UBX PF00789.10 10 UBX domain
AA_permease PF00324.10 −120.8 Amino acid permease
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
FAD_binding_4 PF01565.12 −8.1 FAD binding domain
PALP PF00291.14 −70 Pyridoxal-phosphate dependent enzyme
GIDA PF01134.11 −226.7 Glucose inhibited division protein A
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox PF00070.17 5 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox_dim PF02852.12 −13 Pyridine nucleotide-disulphide
oxidoreductase, dimerisation domain
YGGT PF02325.7 0 YGGT family
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
PEPCK_ATP PF01293.10 −327 Phosphoenolpyruvate carboxykinase
Trp_syntA PF00290.11 −149.8 Tryptophan synthase alpha chain
DapB_N PF01113.10 −20.7 Dihydrodipicolinate reductase, N-terminus
DapB_C PF05173.3 0.5 Dihydrodipicolinate reductase, C-terminus
Ribul_P_3_epim PF00834.8 −97.3 Ribulose-phosphate 3 epimerase family
OMPdecase PF00215.13 −47.3 Orotidine 5′-phosphate decarboxylase/
HUMPS family
Arginase PF00491.11 −120 Arginase family
Pkinase PF00069.14 −70.8 Protein kinase domain
Ferric_reduct PF01794.8 −7 Ferric reductase like transmembrane
component
Lipase_3 PF01764.14 −8 Lipase (class 3)
Auxin_inducible PF02519.4 −15 Auxin responsive protein
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
Anti-silence PF04729.4 25 Anti-silencing protein, ASF1-like
p450 PF00067.11 −105 Cytochrome P450
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
TCTP PF00838.7 −70.7 Translationally controlled tumour protein
NAPRTase PF04095.5 −88.5 Nicotinate phosphoribosyltransferase
(NAPRTase) family
SPX PF03105.9 −20 SPX domain
EXS PF03124.4 20 EXS family
DUF1639 PF07797.3 25 Protein of unknown function (DUF1639)
Mlo PF03094.5 −263 Mlo family
Sulfotransfer_1 PF00685.16 −53.1 Sulfotransferase domain
PI-PLC-X PF00388.8 18.8 Phosphatidylinositol-specific phospholipase
C, X domain
PI-PLC-Y PF00387.8 −11 Phosphatidylinositol-specific phospholipase
C, Y domain
C2 PF00168.18 3.7 C2 domain
HMA PF00403.14 17.4 Heavy-metal-associated domain
Ammonium_transp PF00909.10 −144 Ammonium Transporter Family
Asp PF00026.13 −186.1 Eukaryotic aspartyl protease
SapB_2 PF03489.6 10.7 Saposin-like type B, region 2
SapB_1 PF05184.4 3 Saposin-like type B, region 1
Oxidored_FMN PF00724.9 −147.7 NADH: flavin oxidoreductase/NADH oxidase
family
Pyr_redox PF00070.17 5 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
Pkinase PF00069.14 −70.8 Protein kinase domain
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
Sterol_desat PF01598.7 −13 Sterol desaturase
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
Sulfotransfer_1 PF00685.16 −53.1 Sulfotransferase domain
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
GATase_2 PF00310.10 −106.2 Glutamine amidotransferases class-II
SIS PF01380.11 0 SIS domain
SIS PF01380.11 0 SIS domain
Acetyltransf_1 PF00583.13 18.6 Acetyltransferase (GNAT) family
DUF1005 PF06219.2 25 Protein of unknown function (DUF1005)
DUF231 PF03005.5 −58 Arabidopsis proteins of unknown function
TB2_DP1_HVA22 PF03134.9 −25.1 TB2/DP1, HVA22 family
Di19 PF05605.2 25 Drought induced 19 protein (Di19)
MtN3_slv PF03083.5 −0.8 MtN3/saliva family
MtN3_slv PF03083.5 −0.8 MtN3/saliva family
Pkinase PF00069.14 −70.8 Protein kinase domain
p450 PF00067.11 −105 Cytochrome P450
PGI PF00342.8 −168.9 Phosphoglucose isomerase
Glyco_hydro_28 PF00295.7 −97 Glycosyl hydrolases family 28
Pkinase PF00069.14 −70.8 Protein kinase domain
Isoamylase_N PF02922.7 −6.5 Isoamylase N-terminal domain
Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain
PBP PF01161.9 −20.6 Phosphatidylethanolamine-binding protein
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter
protein
Smr PF01713.11 10 Smr domain
2-Hacid_dh PF00389.19 11.2 D-isomer specific 2-hydroxyacid
dehydrogenase, catalytic domain
2-Hacid_dh_C PF02826.7 −82.2 D-isomer specific 2-hydroxyacid
dehydrogenase, NAD binding domain
DUF1070 PF06376.2 25 Protein of unknown function (DUF1070)
AMP-binding PF00501.16 0 AMP-binding enzyme
Enolase_N PF03952.6 −3.3 Enolase, N-terminal domain
Enolase_C PF00113.12 −34 Enolase, C-terminal TIM barrel domain
FAE_3-kCoA_syn1 PF07168.2 25 Fatty acid elongase 3-ketoacyl-CoA synthase 1
efhand PF00036.20 17.5 EF hand
Na_Ca_ex PF01699.12 25 Sodium/calcium exchanger protein
NOP5NT PF08156.2 25 NOP5NT (NUC127) domain
NOSIC PF08060.2 25 NOSIC (NUC001) domain
Nop PF01798.6 25 Putative snoRNA binding domain
Terpene_synth PF01397.10 −86 Terpene synthase, N-terminal domain
PCI PF01399.15 25 PCI domain
Abhydrolase_3 PF07859.2 25.8 alpha/beta hydrolase fold
GRP PF07172.1 16.8 Glycine rich protein family
DUF914 PF06027.2 −193 Eukaryotic protein of unknown function
(DUF914)
DUF1325 PF07039.1 25 Protein of unknown function (DUF1325)
zf-A20 PF01754.6 25 A20-like zinc finger
zf-AN1 PF01428.6 0 AN1-like Zinc finger
PALP PF00291.14 −70 Pyridoxal-phosphate dependent enzyme
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
DUF1723 PF08330.1 17 Protein of unknown function (DUF1723)
GDPD PF03009.7 −18 Glycerophosphoryl diester phosphodiesterase
family
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
Pribosyltran PF00156.15 2 Phosphoribosyl transferase domain
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
BRAP2 PF07576.1 25 BRCA1-associated protein 2
zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)
zf-UBP PF02148.8 25 Zn-finger in ubiquitin-hydrolases and other
protein
DnaJ PF00226.19 −8 DnaJ domain
HSCB_C PF07743.4 −7 HSCB C-terminal oligomerisation domain
ABC_tran PF00005.15 9.5 ABC transporter
Acyl-CoA_dh_N PF02771.7 10.9 Acyl-CoA dehydrogenase, N-terminal domain
Acyl-CoA_dh_M PF02770.9 25 Acyl-CoA dehydrogenase, middle domain
Acyl-CoA_dh_1 PF00441.13 −15.6 Acyl-CoA dehydrogenase, C-terminal domain
Acyl-CoA_dh_2 PF08028.1 −16.9 Acyl-CoA dehydrogenase, C-terminal domain
NTP_transferase PF00483.12 −90.5 Nucleotidyl transferase
MannoseP_isomer PF01050.8 −70 Mannose-6-phosphate isomerase
Cupin_2 PF07883.1 16.6 Cupin domain
Lectin_legB PF00139.10 −110.1 Legume lectin domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
PGAM PF00300.12 −3 Phosphoglycerate mutase family
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain
PTPA PF03095.4 −106 Phosphotyrosyl phosphate activator (PTPA)
protein
Sedlin_N PF04628.2 25 Sedlin, N-terminal conserved region
NAD_binding_2 PF03446.4 −63.5 NAD binding domain of 6-phosphogluconate
dehydrogenase
6PGD PF00393.8 −232.3 6-phosphogluconate dehydrogenase, C-
terminal domain
ADK PF00406.11 24.2 Adenylate kinase
ADK_lid PF05191.3 25 Adenylate kinase, active site lid
DUF26 PF01657.7 0 Domain of unknown function DUF26
DUF26 PF01657.7 0 Domain of unknown function DUF26
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
mTERF PF02536.4 −60 mTERF
B56 PF01603.9 −210 Protein phosphatase 2A regulatory B subunit
(B56 family)
zf-AN1 PF01428.6 0 AN1-like Zinc finger
Xan_ur_permease PF00860.10 −151.2 Permease family
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
Auxin_inducible PF02519.4 −15 Auxin responsive protein
Trehalose_PPase PF02358.6 −49.4 Trehalose-phosphatase
DUF260 PF03195.4 0.8 Protein of unknown function DUF260
Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
La PF05383.5 25 La domain
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
LEA_2 PF03168.3 25 Late embryogenesis abundant protein
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
DUF241 PF03087.4 −53.6 Arabidopsis protein of unknown function
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
Ion_trans_2 PF07885.4 24.9 Ion channel
Ion_trans_2 PF07885.4 24.9 Ion channel
PI-PLC-X PF00388.8 18.8 Phosphatidylinositol-specific phospholipase
C, X domain
C2 PF00168.18 3.7 C2 domain
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
RNA_pol_Rpb8 PF03870.5 −31.2 RNA polymerase Rpb8
COX5C PF05799.1 25 Cytochrome c oxidase subunit Vc (COX5C)
Ion_trans_2 PF07885.4 24.9 Ion channel
Ion_trans_2 PF07885.4 24.9 Ion channel
Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Na_Ca_ex PF01699.12 25 Sodium/calcium exchanger protein
Na_Ca_ex PF01699.12 25 Sodium/calcium exchanger protein
Pkinase PF00069.14 −70.8 Protein kinase domain
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
OPT PF03169.6 −238.6 OPT oligopeptide transporter protein
PMEI PF04043.5 25 Plant invertase/pectin methylesterase
inhibitor
K_trans PF02705.6 −482 K+ potassium transporter
Pkinase PF00069.14 −70.8 Protein kinase domain
CH PF00307.19 22.5 Calponin homology (CH) domain
EB1 PF03271.6 25 EB1-like C-terminal motif
Carboxyl_trans PF01039.11 −262.3 Carboxyl transferase domain
CoA_trans PF01144.12 25 Coenzyme A transferase
Pkinase PF00069.14 −70.8 Protein kinase domain
CS PF04969.5 8.6 CS domain
SGS PF05002.5 5.2 SGS domain
NPH3 PF03000.4 25 NPH3 family
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Spermine_synth PF01564.6 −93.8 Spermine/spermidine synthase
FeThRed_A PF02941.5 25 Ferredoxin thioredoxin reductase variable
alpha chain
Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain
Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain
Cyclin_C PF02984.8 −13 Cyclin, C-terminal domain
F-box PF00646.21 13.6 F-box domain
F-box PF00646.21 13.6 F-box domain
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
MIP PF00230.9 −62 Major intrinsic protein
LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminal domain
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
WD40 PF00400.20 21.5 WD domain, G-beta repeat
Glycolytic PF00274.9 −174.5 Fructose-bisphosphate aldolase class-I
PSI_PsaF PF02507.5 25 Photosystem I reaction centre subunit III
LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminal domain
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
C2 PF00168.18 3.7 C2 domain
p450 PF00067.11 −105 Cytochrome P450
FH2 PF02181.13 −98.3 Formin Homology 2 Domain
U-box PF04564.5 10.5 U-box domain
FAE1_CUT1_RppA PF08392.1 −192.7 FAE1/Type III polyketide synthase-like protein
Chal_sti_synt_C PF02797.5 −6.1 Chalcone and stilbene synthases, C-terminal
domain
ACP_syn_III_C PF08541.1 −24.4 3-Oxoacyl-[acyl-carrier-protein (ACP)]
synthase III C terminal
aPHC PF05875.2 25 Alkaline phytoceramidase (aPHC)
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Glyco_hydro_14 PF01373.7 −231.4 Glycosyl hydrolase family 14
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
PGM_PMM_I PF02878.5 −37.5 Phosphoglucomutase/phosphomannomutase,
alpha/beta/alpha domain I
PGM_PMM_II PF02879.5 −20 Phosphoglucomutase/phosphomannomutase,
alpha/beta/alpha domain II
PGM_PMM_III PF02880.5 −7.8 Phosphoglucomutase/phosphomannomutase,
alpha/beta/alpha domain III
PGM_PMM_IV PF00408.9 −6 Phosphoglucomutase/phosphomannomutase,
C-terminal domain
Glutaminase PF04960.5 −143.6 Glutaminase
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
2OG-Fell_Oxy PF03171.9 11.5 2OG-Fe(II) oxygenase superfamily
Histone PF00125.13 17.4 Core histone H2A/H2B/H3/H4
F-box PF00646.21 13.6 F-box domain
Agenet PF05641.2 6.6 Agenet domain
Agenet PF05641.2 6.6 Agenet domain
DnaJ PF00226.19 −8 DnaJ domain
SNARE PF05739.8 20.8 SNARE domain
adh_short PF00106.14 −17 short chain dehydrogenase
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox PF00070.17 5 Pyridine nucleotide-disulphide oxidoreductase
NPH3 PF03000.4 25 NPH3 family
PB1 PF00564.13 12.3 PB1 domain
MIP PF00230.9 −62 Major intrinsic protein
F-box PF00646.21 13.6 F-box domain
LRR_2 PF07723.2 6 Leucine Rich Repeat
F-box PF00646.21 13.6 F-box domain
MIP PF00230.9 −62 Major intrinsic protein
Methyltransf_6 PF03737.5 25 Demethylmenaquinone methyltransferase
zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)
RRM_1 PF00076.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or
RNP domain)
zf-CCHC PF00098.12 17.9 Zinc knuckle
zf-CCHC PF00098.12 17.9 Zinc knuckle
NPH3 PF03000.4 25 NPH3 family
p450 PF00067.11 −105 Cytochrome P450
p450 PF00067.11 −105 Cytochrome P450
DPBB_1 PF03330.7 5.3 Rare lipoprotein A (RlpA)-like double-psi
beta-barrel
Pollen_allerg_1 PF01357.10 17.2 Pollen allergen
p450 PF00067.11 −105 Cytochrome P450
p450 PF00067.11 −105 Cytochrome P450
CBS PF00571.16 17.5 CBS domain pair
CBS PF00571.16 17.5 CBS domain pair
Cys_Met_Meta_PP PF01053.9 −278.4 Cys/Met metabolism PLP-dependent enzyme
Beta_elim_lyase PF01212.10 −114.4 Beta-eliminating lyase
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
PK PF00224.10 −244 Pyruvate kinase, barrel domain
PK_C PF02887.5 −44 Pyruvate kinase, alpha/beta domain
PEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain
iPGM_N PF06415.3 −263.4 BPG-independent PGAM N-terminus
(iPGM_N)
Metalloenzyme PF01676.7 −14.4 Metalloenzyme superfamily
Molybdop_Fe4S4 PF04879.5 13.6 Molybdopterin oxidoreductase Fe4S4 domain
Molybdopterin PF00384.11 −50 Molybdopterin oxidoreductase
Molydop_binding PF01568.10 1.1 Molydopterin dinucleotide binding domain
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
HMA PF00403.14 17.4 Heavy-metal-associated domain
DAO PF01266.12 −35.9 FAD dependent oxidoreductase
Pyr_redox_2 PF07992.3 −20 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox PF00070.17 5 Pyridine nucleotide-disulphide oxidoreductase
Pyr_redox_dim PF02852.12 −13 Pyridine nucleotide-disulphide
oxidoreductase, dimerisation domain
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
Hist_deacetyl PF00850.9 −71 Histone deacetylase domain
zf-A20 PF01754.6 25 A20-like zinc finger
zf-AN1 PF01428.6 0 AN1-like Zinc finger
U-box PF04564.5 10.5 U-box domain
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
Arm PF00514.11 17 Armadillo/beta-catenin-like repeat
PDT PF00800.8 25 Prephenate dehydratase
ACT PF01842.13 0 ACT domain
PP2C PF00481.11 −44 Protein phosphatase 2C
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
DUF568 PF04526.3 25 Protein of unknown function (DUF568)
PHD PF00628.17 25.9 PHD-finger
TBC PF00566.8 −58 TBC domain
DUF786 PF05646.3 −31.3 Protein of unknown function (DUF786)
PLAC8 PF04749.6 −1.1 PLAC8 family
IF2_N PF04760.6 25 Translation initiation factor IF-2, N-terminal
region
GTP_EFTU PF00009.15 8 Elongation factor Tu GTP binding domain
Ras PF00071.11 −69.9 Ras family
GTP_EFTU_D2 PF03144.14 25 Elongation factor Tu domain 2
PC4 PF02229.5 4 Transcriptional Coactivator p15 (PC4)
DUF588 PF04535.2 25 Domain of unknown function (DUF588)
LisH PF08513.1 20.7 LisH
CDI PF02234.8 17 Cyclin-dependent kinase inhibitor
Glyco_transf_8 PF01501.9 −43.2 Glycosyl transferase family 8
Pkinase PF00069.14 −70.8 Protein kinase domain
efhand PF00036.20 17.5 EF hand
efhand PF00036.20 17.5 EF hand
efhand PF00036.20 17.5 EF hand
efhand PF00036.20 17.5 EF hand
WWE PF02825.9 25 WWE domain
FAE1_CUT1_RppA PF08392.1 −192.7 FAE1/Type III polyketide synthase-like protein
Chal_sti_synt_C PF02797.5 −6.1 Chalcone and stilbene synthases, C-terminal
domain
ACP_syn_III_C PF08541.1 −24.4 3-Oxoacyl-[acyl-carrier-protein (ACP)]
synthase III C terminal
Hrf1 PF03878.5 −81.2 Hrf1 family
Yip1 PF04893.6 −6.4 Yip1 domain
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
Miro PF08477.1 28 Miro-like protein
Ras PF00071.11 −69.9 Ras family
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
RuBisCO_large_N PF02788.5 25 Ribulose bisphosphate carboxylase large
chain, N-terminal domain
RuBisCO_large PF00016.9 −76 Ribulose bisphosphate carboxylase large
chain, catalytic domain
Flavodoxin_1 PF00258.14 6.3 Flavodoxin
FAD_binding_1 PF00667.9 −79 FAD binding domain
NAD_binding_1 PF00175.10 −3.9 Oxidoreductase NAD-binding domain
Lactamase_B PF00753.16 24.6 Metallo-beta-lactamase superfamily
PGI PF00342.8 −168.9 Phosphoglucose isomerase
Peptidase_C12 PF01088.10 −91.4 Ubiquitin carboxyl-terminal hydrolase, family 1
Dicty_CAR PF05462.2 −39.7 Slime mold cyclic AMP receptor
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
PfkB PF00294.13 −67.8 pfkB family carbohydrate kinase
Brix PF04427.7 11.4 Brix domain
MGS PF02142.11 3 MGS-like domain
AICARFT_IMPCHas PF01808.8 −98 AICARFT/IMPCHase bienzyme
MFAP1_C PF06991.1 25 Micro-fibrillar-associated protein 1 C-terminus
Nicastrin PF05450.4 −85.8 Nicastrin
Ribophorin_I PF04597.4 −217 Ribophorin I
DUF662 PF04949.2 25 Family of unknown function (DUF662)
ARD PF03079.4 25 ARD/ARD′ family
Cupin_2 PF07883.1 16.6 Cupin domain
HATPase_c PF02518.14 22.4 Histidine kinase-, DNA gyrase B-, and
HSP90-like ATPase
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Pkinase PF00069.14 −70.8 Protein kinase domain
Example 5 A
This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
Elements of an exemplary common expression vector, pMON82060 are illustrated in Table 18. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art. The DNA of interest are inserted in a expression vector at the insertion site between the intron 1 of rice act 1 gene and the termination sequence of PinII gene.
TABLE 18
pMON82060
Coordinates
of SEQ ID
function name annotation NO: 33636
Agro B-AGRtu.right border Agro right border sequence, essential for 5235-5591
transformation transfer of T-DNA.
Gene of P-Os.Act1 Promoter from the rice actin gene act1. 5609-7009
interest plant L-Os.Act1 Leader (first exon) from the rice actin 1
expression gene.
cassette I-Os.Act1 First intron and flanking UTR exon
sequences from the rice actin 1 gene
insertion site
T-St.Pis4 The 3′ non-translated region of the 7084-8026
potato proteinase inhibitor II gene which
functions to direct polyadenylation of the
mRNA
Plant P-CaMV.35S CaMV 35S promoter 8075-8398
selectable L-CaMV.35S 5′ UTR from the 35S RNA of CaMV
marker CR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226
expression resistance to neomycin and kanamycin
cassette T-AGRtu.nos A 3′ non-translated region of the 9255-9507
nopaline synthase gene of
Agrobacterium tumefaciens Ti plasmid
which functions to direct
polyadenylation of the mRNA..
Agro B-AGRtu.left border Agro left border sequence, essential for  39-480
transformation transfer of T-DNA.
Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication from 567-963
in E. coli plasmid RK2.
CR-Ec.rop Coding region for repressor of primer 2472-2663
from the ColE1 plasmid. Expression of
this gene product interferes with primer
binding at the origin of replication,
keeping plasmid copy number low.
OR-Ec.ori-ColE1 The minimal origin of replication from 3091-3679
the E. coli plasmid ColE1.
P-Ec.aadA-SPC/STR promoter for Tn7 adenylyltransferase 4210-4251
(AAD(3″))
CR-Ec.aadA- Coding region for Tn7 4252-5040
SPC/STR adenylyltransferase (AAD(3″))
conferring spectinomycin and
streptomycin resistance.
T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 5041-5098
(AAD(3″)) gene of E. coli.
Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean transformation base vector illustrated in FIG. 3 was assembled using the technology known in the art. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.
TABLE 19
pMON82053
Coordinates of SEQ ID
function name annotation NO: 33637
Agro B-AGRtu.left border Agro left border 6144-6585
transforamtion sequence, essential for
transfer of T-DNA.
Plant P-At.Act7 Promoter from the 6624-7861
selectable arabidopsis actin 7 gene
marker L-At.Act7 5′UTR of Arabidopsis
expression Act7 gene
cassette I-At.Act7 Intron from the
Arabidopsis actin7 gene
TS-At.ShkG-CTP2 Transit peptide region of 7864-8091
Arabidopsis EPSPS
CR-AGRtu.aroA- Synthetic CP4 coding 8092-9459
CP4.nno_At region with dicot
preferred codon usage.
T-AGRtu.nos A 3′ non-translated region 9466-9718
of the nopaline synthase
gene of Agrobacterium
tumefaciens Ti plasmid
which functions to direct
polyadenylation of the
mRNA.
Gene of P-CaMV.35S-enh Promoter for 35S RNA  1-613
interest from CaMV containing a
expression duplication of the −90 to −350
cassette region.
insertion site
T-Gb.E6-3b 3′ untranslated region  688-1002
from the fiber protein E6
gene of sea-island cotton;
Agro B-AGRtu.right border Agro right border 1033-1389
transformation sequence, essential for
transfer of T-DNA.
Maintenance OR-Ec.oriV-RK2 The vegetative origin of 5661-6057
in E. coli replication from plasmid
RK2.
CR-Ec.rop Coding region for 3961-4152
repressor of primer from
the ColE1 plasmid.
Expression of this gene
product interferes with
primer binding at the
origin of replication,
keeping plasmid copy
number low.
OR-Ec.ori-ColE1 The minimal origin of 2945-3533
replication from the E. coli
plasmid ColE1.
P-Ec.aadA-SPC/STR romoter for Tn7 2373-2414
adenylyltransferase
(AAD(3″))
CR-Ec.aadA- Coding region for Tn7 1584-2372
SPC/STR adenylyltransferase
(AAD(3″)) conferring
spectinomycin and
streptomycin resistance.
T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 1526-1583
adenylyltransferase
(AAD(3″)) gene of E. coli.
Example 5 B
This example illustrates monocot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation of corn callus. Corn plants of a readily transformable line are grown in the greenhouse and ears harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryos are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes. Immature embryos are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformants are recovered 6 to 8 weeks after initiation of selection.
Plasmid vectors are prepared essentially as described in Example 5 for transforming into corn callus each of DNA identified in Table 1 and the corresponding DNA for the identified homologous genes identified in Table 2, by Agrobacterium-mediated transformation.
For Agrobacterium-mediated transformation of corn callus, immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media, and paromomycin resistant callus identified as containing the recombinant DNA in an expression cassette.
Transgenic corn plants are regenerated from transgenic callus resulting from transformation on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber at 26 degrees C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g. for yield trials in the screens indicated above. Populations of transgenic plants and seeds produced form transgenic plant cells from each transgenic event are screened as described in Example 7 below to identify the members of the population having the enhanced trait.
Example 6
This example illustrates dicot plant transformation to produce nuclei of this transgenic in cells of transgenic plants by transformation of soybean tissue. For Agrobacterium-mediated transformation, soybean seeds are germinated overnight and the meristem explants excised. The meristems and the explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed germination and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Trait positive shoots are harvested approximately 6-8 weeks post bombardment and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil. Populations of transgenic plants and seeds produced form transgenic plant cells from each transgenic event are screened as described in Example 7 below to identify the members of the population having the enhanced trait.
Example 7
This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Populations of transgenic seed and plants prepared in Examples 5 and 6 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait. Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below. Plant cell nuclei having recombinant DNA with each of the genes identified in Table 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.
Selection for Enhanced Nitrogen Use Efficiency
Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed before planting by collecting sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.
Transgenic corn plants prepared in Example 5 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention. Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention. Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention.
Selection for Increased Yield
Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform. well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre.
Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 5 and 6 are screened for yield enhancement. At least one event from each of the corn and soybean plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.
Selection for Enhanced Water Use Efficiency (WUE)
The following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/− ˜one day for each step given the situation.
To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants of this age run around 98% RWC.
Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for water use efficiency. Transgenic plants having at least a 1% increase in GRG and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance improvement (DS) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.
Selection for Growth Under Cold Stress
Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C. for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty millilters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
The germination index is calculated as per:
Germination index=(Σ([T+1−n i ]*[P i −P i-1]))/T
where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.
Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for water use efficiency. Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance improvement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.
Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels
The following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
Infratec® Model 1221, 1225, or 1227 analyzer with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.
TABLE 21
Typical sample(s): Whole grain corn and soybean seeds
Analytical time to run method: Less than 0.75 min per sample
Total elapsed time per run: 1.5 minute per sample
Typical and minimum sample Corn typical: 50 cc; minimum 30 cc
size: Soybean typical: 50 cc; minimum 5 cc
Typical analytical range: Determined in part by the specific
calibration.
Corn - moisture 5-15%, oil 5-20%,
protein 5-30%, starch 50-75%, and
density 1.0-1.3%.
Soybean - moisture 5-15%, oil
15-25%, and protein 35-50%.

Transgenic corn plants and soybean plants prepared in Examples 5 and 6 are screened for increased protein and oil in seed. Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.
Example 8
This example illustrates monocot and dicot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant DNA suppresses the expression of an endogenous protein identified by Pfam, Histone, WD40, NPH3, FHA, PB1, ADH_zinc_N, NAPRTase, ADK_lid, p450, B56, DUF231, C2, DUF568, WD40, F-box, Pkinase, or Terpene_synth. Corn callus and soybean tissue are transformed as describe in Examples 5 and 6 using recombinant DNA in the nucleus with DNA that transcribes to RNA that forms double-stranded RNA targeted to an endogenous gene with DNA encoding the protein. The genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQ ID NO: 426, 428, 429, 430, 524, 525, 541, 601, 602, 650, 651, 654, 655, 657, 660, 694, 698, 772, 801.
Populations of transgenic corn plants and soybean plants prepared in Examples 5 and 6 with DNA for suppressing a gene identified in Table 3 as providing an enhanced trait by gene suppression are screened to identify an event from those plants with a nucleus of the invention by selecting the trait identified in this specification.
TABLE 2
SEQ ID NO: homolog SEQ ID NOs
426: 31549 26664 26645 25553 29273 18258 31741 1551 3388 3387 3064 3017
3389 3999 3063 3998 3335 1921 2854 2472 3997 2355 2445 2444
2706 2471 3248 2807 32559 10182 33093 29066 22058 29063 20705 31709
6002 5864 6338 5023 4626 5024 6337 6427 5000 5459 5995 4979
6428 6010 6045 4493 6336 4977 5797 4981 4976 5620 4492 5022
4247 5044 5006 6046 6339 5629 6208 6044 5458 5578 5460 5730
5025 6335 5127 4630 5836 6207 6334 4540 5837 6383 5119 5031
10181 4764 5504 6014 5211 4500 5835 6384 6104 4571 4765 5788
4499 5778 6364 6357 6233 4501 6361 5777 4665 6231 4309 6356
6234 4842 4115 4268 5392 6239 6012 4135 6232 5765 4198 5796
4898 4896 4495 4174 4126 6230 5013 4498 10574 6382 6237 6362
6238 5795 4766 6236 5040 4897 4494 5012 6089 6360 5038 8703
9296 7544 7284 9368 7945 9335 8704 7048 9329 7543 9353 8306
7545 7944 8345 8735 27146 8792 7422 7390 7777 7719 9354 7311
7776 7943 7718 9391 8921 7556 8687 8137 7282 8775 9334 10206
33181 25349 28507 29674 12747 13519 17862 18456 33494 19906 26998 26997
20607 20608
427: 25164 21072 28215 30323 31072 1539 2857 2023 3862 3765 30194 4603
4849 4874 6309 4873 5370 7588 8285 6789 7438 8158 16525 21509
31609
428: 23535 26657 26659 25656 22696 1550 1502 1035 3630 3084 2821 2820
3631 3491 3281 28996 5526 5544 5212 5600 6215 4590 5721 5483
7411 7413 7087 6763 7285 8215 7831 8475 7336 7665 7830 9360
8005 6817 19924 23111 13730 29173 20696 18769 16648 24623
429: 1297 1585
430: 1005 893 1155 1664 1798 1351 1677 2081 22028 28867 7193 8619
7005 8839 32857
431: 25541 1508 1509 1553 3042 3874 2425 3380 2438 2089 2165 31105
22015 4633 5715 6165 5109 5104 6018 4457 5002 5709 8234 8233
8887 6593 9390 20693
432: 17645 32929 16109 16115 29937 17418 12553 24238 11719 13895 18572 17067
15142 14984 12937 32611 17020 28098 21188 16710 27572 11897 20037 20035
20036 20034 17111 18055 28529 19049 18104 18105 13984 19500 25953 20452
20014 20969 20555 30341 22310 33377 21605 21382 21278 24058 25799 24665
25099 30029 27722 25477 10712 25303 26450 26343 12378 16213 16083 12602
12023 18632 28013 27361 28132 20347 13710 30404 29726 1027 1629 11636
1950 2653 2550 2419 3624 2523 2999 3403 14341 11278 29058 5150
6157 6259 5151 5083 6272 7653 6935 8821 8308 7111 8992 6561
8750 6934 8702 8822 6613 32327 33273 28685 10183 22836 30888 15219
29975 30401 30402 30403 16604 12184 33248
433: 22629 1018 1684 928 1084 10512 3923 3013 3941 2520 4022 3940
3543 2522 3352 2521 21983 29335 29050 29049 14413 4107 7164 8885
6641 6682 8193 7548 13731
434: 1467 2508 3970 7052
435: 1255 1124 958 1253 1254 1501 1549 1072 2612 3537 2443 3701
4062 2663 3374 1932 2828 1951 6185 5932 4263 4723 6434 5884
5933 4604 4593 4374 5240 5639 5221 9062 7723 6978 6874 9185
7700 7668 6650 7929 6857 6992 8404 7791 6931 6564 6728 13457
436: 11330 11798 11797 11803 11800 11802 11796 11807 11799 11801 11806 11805
11804 11808 24821 1221 22575 3994 2950 2357 3993 13245 17749 32992
33068 18403 16916 31336 7187 17867 27005 20112
437: 1606 3877 3448 3876 2580 3552 2836 3074 2181 3433 3509 2498
29402 22027 5283 5447 8053 7217 9237 8787 9236
438: 11753 17885 1367 22014 19360 5817 6826 8128
439: 14793 15421 1784 886 1769 1620 1147 1676 1349 1130 1555 880
29150 21099 5008 6469 9122 7083
440: 1793 1191 2507 3786 3326 2547 2895 3788 3787 5050 4592 4302
5051 5049 8723 7236 8412 8882 7135
441: 1017 1131 1696 3498 2936 2897 2288 3478 1846 28866 28999 5982
4505 6279 4635 6217 5182 4846 8930 6829 8264 8123 6872 8586
8585
442: 33099 13572 30102 25061 13115 32911 32896 11209 17364 25755 13934 14599
14606 17072 15091 17542 23794 23810 15791 16272 27533 16826 20491 20504
11964 11932 17180 17150 16340 24750 18956 12797 12083 13988 25948 25961
30124 30339 22859 21343 28274 30165 24966 25262 31065 31064 27926 27216
9467 30676 26106 28365 28321 28633 3237 3011 3098 29342 29229 4100
8573 8809 8808 8810 28776 31168 31178 21744 31417 13203 32848 24632
443: 1495 1430 1106 1631 3776 3775 3049 2841 3001 2344 2981 3315
2494 3330 3249 21974 31098 5684 5069 5661 4632 5068 7489 6894
7840 7889 7490 7628 8528 8518 6686 14775
444: 23594 24432 26726 25623 22755 19732 16724 32850 1685 1129 964 1159
9579 29036 27426 5582 5224 5225 4867 4314 4266 5226 4265 4866
5714 5354 8768 9203 7166 8133 7519 7521 7619 7855 7691 8960
7520 6579 7658 7051 8900 7370 9823 33308 29175 20627 2220 3390
3971 3972
445: 21160 23464 24408 24537 24340 26791 26667 26756 25617 25632 25636 25653
22643 22765 22766 22900 9795 23354 32042 32047 28015 28340 16640 24622
19287 1789 31335 32423 32422 19273 20734 29107 32424 19902 10176 32983
20086 15242 15371 23679 11429 23746 17863 17864 5776 4405 6425 7555
7260 6966 7614 9227 7115 8828 9226 7613 31959 33318 11328 10868
13606 33018 13605 22972 20711 14417 16765
446: 28240 10652 26605 29336 29337 24953 26604 31901 15972 22181 26907 14237
21168 21170 21169 13227 22097 27430 32943 30651 30652 31646 33594 31648
31649 32094 32935 31922 20728 20079 972 31696 32640 32638 10064 32643
32644 15930 15931 22550 28927 28945 28946 28916 21098 26524 18630 31654
18631 32536 31281 11270 14312 11269 18402 31645 31644 23663 10224 18437
13974 17977 4647 5686 6222 5118 5188 9061 6568 8101 8170 13782
13781 13783 10797 10798 13286 26992 16652 31911 14189 22105 13447 9969
14573 15981 25196 10932 10931 10933 10930 10934 31802 15396 33325 24598
32967 14771 14228 33323 33324 20365 33262 20136 18760 33322
447: 22160 15597 15598 1245 1163 1632 1600 1614 1324 1302 21532 24130
3649 25355 9913 25356 22119 32161 31123 25399 24030 32165 32164 14822
17694 6514 18246 19892
448: 1732 1295 2927 3709 2305 2182 3421 3295 3599 2051 1866 3686
3562 3569 3320 2284 5823 6340 5801 6290 6483 5770 8608 8490
7061 25361 32572
449: 1069 1107 3644 2886 3353 2506 3160 3459 31109 26533 4645 6316
5915 4363 7014 7015 6973 6916 9089 8537 7510 6974 7085 6862
15738
450: 10652 26605 29336 29337 26608 24953 26604 31901 15972 22181 14237 21170
21168 21169 13227 22097 27430 32943 17894 30651 15348 31646 33594 31648
31649 32094 32935 31922 20728 20079 32638 10064 32643 32644 15931 15930
22550 28916 21942 20838 28957 29060 28927 29027 28946 29261 28945 29260
29035 21098 26524 18630 31654 18631 32536 31281 14312 11269 11270 18402
31645 31644 10224 18437 13974 17977 31348 5686 4647 9061 6568 13782
13781 13783 10797 10798 16652 28907 31643 31911 14189 9969 22117 15981
25196 31905 10932 10933 10931 10934 10930 31802 11779 11780 11781
451: 26991 1104 1986 1889 1914 1919 1984 3561 2173 3162 2335 3177
22016 29109 4192 6515 5223 4622 7947 6820 6886 8294 9189 8914
9365 7350 9045 8470 7041 7257 6587
452: 30274 14219 18232 30314 25837 25227 27755 26409 26264 30622 12638 12219
11870 1268 1818 2368 4016 2409 3482 11872 2408 1960 2906 2407
2406 2003 2097 2504 2765 11871 15576 20252 10864 5804 6401 6161
4817 4275 5914 5790 6189 4137 4788 4233 5596 4838 6011 8106
9293 8426 9292 21100 6906 8667 7271 6891 7170 6779 6773 7076
9411 14808 15740 16626 33027 29457 15879 11869
453: 33449 24954 11331 28872 23556 23581 24330 24535 26667 25640 25657 22676
22631 21212 19284 19729 31579 31580 28503 13283 11257 11256 11255 11254
944 977 966 854 1235 1526 1733 31339 31340 29635 31234 3928
2110 3030 2336 24866 10954 31341 31342 22018 31233 18873 31344 31343
31345 10338 11549 5361 5670 4117 4124 6959 7410 6960 7022 8403
32961 9046 8045 10890 9109 9057 8552 20959 32715 22099 22831 18876
20708 13253 17897 10970 10058 31776
454: 1365 3329 3974 2036 3892 2206 20308 5950 4701 5390 5270 6017
8016 6635 9169 7950
455: 14727 27390 33297 25773 21054 9775 22266 31040 9863 32233 10423 20072
9987 14229 15243 1761 1505 1588 971 1706 1037 1128 2673 2100
2603 2860 3986 2168 2941 2198 3037 2989 3385 1852 2686 2991
4092 3094 3058 1936 3069 3035 2007 3004 2148 23007 21946 19900
19773 32394 4127 4409 5411 5407 5269 5405 5410 5409 4677 5393
5408 4316 5387 5412 5133 5400 5406 6184 5610 4293 4470 5733
4836 4294 8539 8924 7846 8246 6630 7102 7847 8434 8245 7716
6625 9007 8615 6785 8516 7845 8628 8545 6603 8501 6602 7988
6758 7100 22114 17748 31591 21491 11867
456: 2302 3412 3861 2660 3197 3019 2037 3043 4010 3150 3127 2301
2883 3327 29014 29126 30207 5813 4420 6188 4488 5339 5973 4242
5520 5986 5697 5675 4845 6772 9145 7475 8414 7242 8694 7427
8713 8901 7895 6937 6795 7262 8318 8317 7345 7346 7896 6559
7380 8949 15891 32860 15739 33026
457: 1560 1290 3228 3733 22906 29032 32023 6024 8630 8579 9012 9554
14431
458: 11386 27460 13968 23583 24335 24456 26772 26698 25546 22719 11788 24040
33254 1790 998 1312 1218 1353 1074 13967 23062 2973 3872 22118
14252 24829 15068 15758 9998 24867 24937 15578 24936 15579 11143 15580
10544 13615 18405 16029 11162 13465 12271 14766 22947 20701 20961 10965
30762 20606 14634
459: 3549 2711 3511 3606 2038 2330 5747 4812 5688 5465 8968 9378
7525
460: 21674 17217 29209 32673 17228 33139 33337 11123 33462 24991 17634 13525
20532 13549 12213 27645 27634 30261 30217 760 30222 30264 30233 31539
25803 30097 10349 13101 11157 13129 31505 32924 32906 17681 18668 11154
10345 21672 33598 21671 14953 23707 19274 14517 18628 17993 27015 19997
23020 17608 15594 10339 26609 10495 24134 14733 10298 10267 10310 10275
10306 16104 16096 16128 16112 27874 27867 11214 11175 11202 14422 29947
25898 24094 31742 17294 17381 17393 17384 17355 12568 12582 24838 24243
24227 30005 25764 21075 21055 26552 13409 13407 19742 18359 18317 18331
18339 12857 13851 13228 14202 14212 14191 18556 18567 14366 14369 14355
14374 27120 15686 15717 15698 15677 17740 17725 14609 14577 14582 23896
17059 17071 32887 14684 14670 15196 15035 32292 31377 32601 15089 15109
15114 15531 15564 20106 14142 17529 17545 22492 22425 15084 23796 32417
15794 17016 16998 17028 30978 19928 15523 17817 17836 17795 17828 17764
28058 16294 16291 29821 28078 28226 18792 16789 16791 16798 26001 16668
16863 16857 32374 32363 27547 27538 27558 16823 29850 16899 20518 29891
29906 11892 11913 11950 19245 19266 19225 19248 17182 17107 17088 17108
18599 12732 18067 18044 18080 28557 28564 28603 28549 28559 28617 18847
11158 18711 18682 16346 16369 16322 16342 24690 24766 18884 18882 18915
18919 18912 18930 19074 19106 19115 19073 12772 33618 481 12047 9873
9903 9895 18120 14033 14029 14021 19336 19792 19872 19814 23590 23504
19508 19584 19688 19616 19580 19676 19568 19596 19665 29925 20483 20466
29913 20464 20017 21927 15746 24996 24390 24490 24498 24361 24463 9678
20976 20983 20563 20562 26653 26741 25544 25607 30137 25038 25891 25878
30116 22728 22779 21720 20787 20783 12468 32195 32209 23179 12350 12342
30342 30362 22344 22303 22355 22338 22371 22305 22361 12315 33409 33430
21663 21613 21806 22290 22227 22258 22219 22856 21429 21434 21480 21347
21342 21351 21330 21397 21263 21258 21280 21222 24029 29479 29489 25914
24070 24065 28261 23924 23933 23958 25170 25739 25729 26055 26057 26078
26059 24653 25867 27490 27510 30085 26601 24291 28289 28285 28272 30026
25131 26960 26958 25667 30153 25691 25681 27796 25437 11680 13046 10742
10744 10685 24973 24960 27102 13771 25277 25278 25261 31047 31518 26489
26499 26423 26394 26356 26260 26185 27930 27945 27935 27973 27974 27296
27272 11362 11368 11375 9474 9512 9538 9470 9504 11497 11468 11508
11463 16454 29780 30632 30630 30602 12384 13330 13328 13334 26095 16217
12504 12518 10370 10372 30505 30525 30513 30515 30510 16054 26032 16042
16061 16070 12640 12636 12238 14942 14938 10816 32463 24104 27984 27382
27320 18003 28352 28408 28385 28140 28496 28455 28428 28477 28325 28313
17574 17432 30767 32130 32128 33561 13649 17626 20531 12488 18504 24025
18461 14447 16439 29704 16449 17586 32129 15398 11548 15412 18015 33475
31347 17579 32864 28644 32280 15941 1060 1416 1019 32038 17665 14880
11623 11574 10628 3546 2891 3736 3678 3899 3165 3913 3083 3612
2837 3912 2225 1835 3920 2738 1871 1870 2733 2767 18393 29285
29399 30025 14567 14566 10463 4770 5738 4145 5515 4913 4637 5189
5635 6138 9044 9206 9408 9197 8857 9242 7800 8859 8858 8856
6684 9239 6575 9811 11814 32980 32670 22102 33033 32112 31913 20617
33336 23708 21004 10207 30746 31908 31906 31907 14409 33276 9965 21553
21551 461 713 28769 28707 28710 28708 28720 23742 28632 16521 16568
16577 12864 12460 31203 31152 31196 31193 31137 20526 32050 27143 31329
31313 31299 20859 20866 20879 20877 20882 21496 21523 30933 22109 12135
12118 31534 12988 12965 28251 10617 21755 21749 21512 21515 16591 12168
26863 26874 31444 31423 31433 31438 27532 29374 13193 13164 13191
461: 21674 17217 32673 24991 33462 29209 33139 17228 21008 11123 17634 13525
12213 27634 27645 27613 30261 30217 760 30222 31539 25803 13101 31505
32924 17681 18668 10345 21672 33598 14953 23707 19274 14517 18628 17993
27015 19997 23020 32981 26609 10495 10267 10298 10275 10310 10306 16104
16128 27874 11214 11175 14422 25898 29947 17294 17381 17384 17393 17355
17391 17298 12568 24838 24227 24243 30005 21075 26552 13409 19742 19744
18359 18317 18331 13851 12855 13948 12857 13936 13228 14202 14212 18556
14366 14355 14369 14374 27120 15686 15717 15698 17740 14609 14577 23896
32887 14684 15196 15035 12875 12909 32292 31377 32601 15089 15109 15531
17529 17545 22492 15084 23796 15285 32417 15794 16998 17016 17028 30978
19928 15523 17817 17795 17836 17828 27894 27896 27897 28058 16294 29821
28078 28226 18792 16789 16798 26001 16668 16863 32374 32363 27547 27538
27558 16823 29850 16899 20518 29891 29906 11892 11950 11913 19245 19225
19266 19248 21877 17182 17107 17088 18599 12732 18067 28557 28564 28603
28549 28617 28559 18847 11158 18711 18682 16346 16369 16342 16322 24690
24766 18884 18882 18915 18919 18930 18912 19074 19106 12772 12047 9873
9903 18120 18149 18154 18182 14033 14021 14029 19336 19792 19872 19814
23590 23504 23621 19508 19584 19688 19580 19676 19683 19563 19616 19596
19568 19665 29925 20483 29913 20017 21927 15746 24996 24490 24498 24390
24361 9678 20976 20983 20425 20563 26653 26741 25544 25607 30137 25891
25038 25878 22779 22728 21720 20783 20787 12468 32195 32209 12473 23179
12350 30342 30362 22344 22303 22355 22338 22371 22361 22305 12315 33409
33430 21663 21613 21806 22290 22258 22227 22856 21429 21434 21456 21480
21342 21347 21351 21304 21329 21397 21258 21263 21280 21225 21261 21222
29479 29489 24070 28261 23924 23933 23958 23983 25739 25170 26055 26057
26078 24653 25867 27490 27510 30085 24291 28289 28285 30026 26960 25667
30153 25691 25681 25678 27796 25437 11680 13046 13038 10742 10744 10659
10685 24973 27110 27055 13771 25277 31047 26489 26423 26394 26356 26260
27930 27945 27935 27973 27296 27272 11362 11368 9474 9512 9504 9538
9470 11497 11468 11508 16454 30632 30602 12384 13330 16217 26095 12504
12518 10370 30505 30525 30513 30515 30510 16054 26032 16061 16070 16042
12640 12636 12635 12238 12228 12244 14942 14938 10816 32463 24104 27984
27382 27377 27320 18003 28352 28408 28385 28140 28496 28455 28428 28325
17574 17432 30767 32130 32128 33561 460 13649 13651 17626 30766 20531
12488 18461 14447 16439 16449 29704 10205 33475 15398 32129 11548 31347
17579 18015 15412 32864 1060 911 1565 32038 17665 14880 11574 11623
10628 3678 3165 3736 3612 2891 2837 3546 3913 3083 3912 1835
2225 3899 3920 2738 2767 18393 22157 29285 30025 29399 28932 29096
10463 4913 5738 4637 5189 4770 5635 4145 5515 6138 5576 5577
9044 9206 9408 6684 9239 6575 9242 9197 8857 8858 7800 8856
8859 7931 9811 11814 22102 32670 32980 32331 33033 9978 32112 31913
20617 33336 23708 21004 30746 31908 31906 31907 14409 33276 9965 21553
28769 28707 28710 28708 28720 23742 28632 16521 16568 12864 12449 31203
31196 31137 31193 31152 20526 32050 27143 31313 31329 31299 20859 20866
20879 20877 20882 21496 30933 11041 12135 12118 12148 31534 12965 12988
28251 10617 21749 21755 21512 26863 31444 31433 31423 27532 13164 13193
13191
462: 11312 31372 20362 14449 33006 27650 33631 13095 10631 31804 10308 10286
30295 29564 11184 24096 17416 17377 12588 29597 19912 13414 24934 18366
13916 14223 16655 18581 14381 14368 14378 15666 14619 14486 15133 14972
12941 9943 32598 15098 15535 15533 14138 14158 14160 17498 17553 17562
17555 22449 22422 23771 23826 23823 17025 17009 19930 17832 17806 17820
27916 28065 16295 16683 26004 16672 27602 16818 16811 29855 29841 16894
29888 12003 11901 19215 17169 17110 17097 18617 18600 18059 18058 18041
28517 28560 16391 18980 19045 19046 19119 12795 12080 18230 14020 14009
19409 19410 19296 23520 19657 19659 24347 9688 20433 26760 26765 25614
22792 21727 20775 32184 23227 30334 22368 12312 24300 33402 33401 21665
22875 21477 21393 21283 23245 25909 25928 23908 25182 30053 28273 26965
25228 27728 25466 25491 10702 10688 27084 27049 26419 26434 26208 26271
11364 9485 11502 11480 11490 16456 16464 29746 16493 12393 13335 16202
26137 26143 16209 16210 12493 30518 30532 30507 26011 16069 14900 14904
14930 10844 28027 27317 10130 28439 11817 24814 13956 23306 13716 29717
31989 33279 20134 14836 1687 14293 11629 11616 2247 3943 2415 3410
3515 2946 3944 3942 3128 3100 3568 9717 31948 31947 22036 14306
23287 4684 5897 8671 7334 9228 7857 6556 8247 7227 8435 6876
10914 11235 10951 31622 19275 33194 31664 14086 28767 31187 31219 31321
31315 31328 31303 31316 20884 20867 30898 12160 12985 12972 12982 14732
21762 480 31540 26864 26853 15228 31426 13192 13180
463: 9801 33260 17216 9587 32516 32172 30219 32674 9944 33355 27012 27830
27842 11819 11445 16961 16963 24833 16010 10337 14738 32809 32034 28337
20212 12492 15932 23352 31680 21089 9774 10302 10300 13624 11178 20930
32561 29586 16132 32986 32001 16136 32987 29583 16150 16142 10909 10897
10888 10902 18667 18666 30406 10938 15346 13452 13438 13366 13380 13372
13369 13370 13365 13439 13384 13359 13393 12664 12643 12649 12680 12646
12653 12704 12687 12691 12716 12689 12694 18262 18345 11723 13931 14549
14536 14526 14540 14352 14365 15708 33019 33023 12905 29640 23087 31516
17984 19035 19022 19034 19018 14154 22419 30976 19937 17822 17790 17842
27923 27910 16234 16250 16233 28115 17697 17698 15907 19262 19246 17203
17204 17195 17193 17473 17463 17469 17250 18038 28544 28619 28620 28555
28520 28562 28524 28618 28593 18733 18732 18681 18749 18756 18735 18748
18750 18696 18705 18741 16382 16331 19114 12771 12775 28911 18184 18124
19390 19381 19398 19373 19370 19371 19411 18521 19862 23557 19567 19632
19639 31909 16958 24546 24519 9708 19715 11443 15870 30350 22342 22309
24314 33388 21609 24190 24176 24178 24177 24211 24188 23143 25186 26172
27508 28824 28846 28834 28838 28823 28825 28845 28826 28839 26987 26926
26938 26929 26932 26930 26925 26988 26919 26948 30148 27798 27083 27091
27093 27101 27094 27095 27079 27075 27074 27034 27033 27030 27047 27050
27044 27273 18257 9490 9536 9459 11470 11488 11521 11479 11495 11455
11496 12365 12375 12386 12396 12401 12395 13358 13293 13306 13295 13298
13296 13292 13288 13315 26115 26093 24589 17915 17914 26017 12239 14898
14897 10832 17696 28155 28169 28198 28193 28166 28028 28358 28485 28432
28319 28296 15648 9573 11428 20346 14407 1006 946 13627 11056 32272
32273 32271 31941 11631 11737 14825 17232 23396 27413 33032 17939 33250
20614 20379 24159 30482 24158 21207 30542 24931 27522 9850 33284 19183
33277 11241 14956 31156 31149 31221 15237 31325 20862 9870 28343 27001
20586 12983 12992 12980 10619 13786 24927 31415 31449 13198 13188
464: 23571 24368 25650 22753 1136 1052 1804 1763 2801 3585 2871 2872
2802 2835 3937 3184 1990 2351 2803 3185 3737 30214 28958 6385
4234 6139 4725 6280 4858 6342 6273 5285 5628 5513 7104 9124
9225 9125 8920 7747 7746 9425 7916 8789 8788 7744 6943 7455
8260 6754 8500 6972 6952 8261 6669 8964 7745 7202 6963 9224
6824 9386 32180 33469 9855 9974 24801 24800 33298 16651
465: 24861 15227 23325 20850 15069 33492 12210 30227 15339 18531 25064 17433
13125 32898 31135 14843 14415 17283 10515 11261 31677 15226 10303 30292
16121 26917 17752 29959 16160 31025 17411 12557 31590 33215 24240 25162
21045 13431 12650 12692 18311 11727 13913 18375 14201 21035 18578 14548
17734 14615 14607 17054 14687 14496 15194 15033 12935 32626 15095 19007
15567 17532 22486 23800 15814 23030 15520 17823 28071 16249 28103 18781
21187 16707 26006 16870 32379 27569 16812 20386 29853 15918 11904 23406
21863 21862 20829 17167 17483 17095 18590 21179 26829 18073 28599 18843
18689 16375 24778 18886 19098 12052 9894 17962 18130 13987 18495 19406
19310 19506 19661 19746 29921 25964 24467 9702 20335 20981 21005 33175
20547 20528 26634 25638 30133 21725 20806 32206 12344 30336 22319 10089
24312 21014 33367 21590 22260 22861 21422 21309 21238 24195 23241 23135
29483 29960 23928 25721 26056 29990 29998 23166 27513 25088 26586 24262
28278 30027 28844 26981 25207 27661 27656 25326 10707 24983 27114 27059
25288 27159 31044 26420 26323 26197 27969 27256 11360 9527 12045 11474
16482 30624 12367 13350 16220 26156 12506 24585 17923 13275 10408 30519
26015 12252 14921 10827 10595 21006 18247 18248 28162 27345 27349 28379
28146 28449 31588 32873 32872 33180 28669 28668 17680 29309 17437 17224
18373 30013 30015 29291 29290 13659 31636 29453 19733 11816 29695 13265
13264 31578 18649 15923 14277 10539 19995 22210 22211 22212 19194 15382
20148 13494 9575 10347 31803 9574 1727 856 1607 1531 1535 857
1608 1328 1756 31586 31587 33052 10540 23323 477 11626 23063 4018
3300 4017 1838 3286 3323 3285 2834 2570 4000 3322 2748 2267
9733 32132 23108 20083 18469 33313 19522 28857 32011 32013 32012 16981
29345 28933 29069 29396 27122 11656 20325 23293 13487 23019 14473 9948
9947 21092 18523 20075 20076 33453 33451 33450 33452 33456 33454 33455
32395 23029 31809 23027 23028 19519 19520 32550 19516 32551 19518 32549
14435 19517 23317 29211 10537 21855 15338 5781 4835 6269 6061 5760
5342 4734 4843 4672 18530 18529 5167 5827 4832 6433 5168 5181
4179 5759 4270 4831 5757 5896 4833 4149 23324 23318 23320 23319
9352 7450 7055 8444 7739 7740 9282 9280 8284 6706 18532 15340
8752 7883 9281 7844 7306 8061 6827 15926 9585 16721 9749 20440
17225 20253 13150 16304 10963 13626 30452 19745 20679 26828 32546 23004
31283 19524 31589 15337 15336 15335 28740 16571 12446 17866 17865 17714
17713 18410 18409 18408 18412 18411 18407 31584 31585 19996 31208 31312
17936 20874 30890 32237 32179 13841 12113 32134 22556 32131 23107 32321
32322 23109 32133 32323 32136 21760 21517 23326 16607 12187 26867 12832
466: 14225 14345 17736 14579 14674 15112 18518 19714 9909 33135 24034 24483
9706 22641 12348 25934 27486 24964 27944 11378 30606 30502 12250 12847
11244 10822 32104 1579 866 1122 1098 1475 22961 3938 2107 3174
3939 3175 29106 24677 20932 5532 5531 4892 4139 6513 9230 7235
7859 32584 18819 33342 20715 19150 20687 20660 19147 10331 10968 12444
31294 20856 28043 33608
467: 24433 1415 1742 3346 2062 3726 2609 3727 3528 2887 3180 3154
2082 2519 2888 3357 3394 3181 2083 4088 1886 1925 3215 2178
5704 6142 4397 5510 5978 4238 4870 5514 4237 9087 6856 9176
8991 7692 8829 6855 9174 8301 7679 8105 7147 8547 9175 7018
8268
468: 5812 4119 5257 7249 8502 8664 8836 8835 8880 6942 9129 14103
469: 29336 29337 32646 32645 26606 26604 31901 28914 14237 21168 21169 21170
22097 27430 30651 12428 30652 28341 15348 33594 31649 31648 32093 32935
31922 20728 1702 1703 1717 1173 1359 3663 32641 32640 32642 32637
32638 32636 17754 32644 32643 32639 22550 29022 23031 26524 32536 31644
31645 23663 18437 31551 31348 28642 6913 8332 13286 26547 11223 31643
31911 14189 13447 22105 9969 31552 22117 15981 25196 10930 10933 10934
10932 10931 29458 32966 15735 15403 33320 9561 19738 23006 33489 24629
27206 17875 24612 20121 17882 24597 33321 29329
470: 2261 3392 2758 2900 3131 32029 5941 5680 4583 6986 7395 26901
471: 30247 24003 1330 1528 912 3247 3015 1942 4086 2456 3345 2994
3014 2457 1880 3099 8963
472: 22414 29412 1730 24942 3910 1922 2632 2555 3613 2556 2899 29016
29104 29000 29200 20294 29252 29134 26906 11275 30394 20078 20254 15374
5990 5907 5803 7128 8911 9397 6994 29207 10480 10481 18862 10875
24600 9804 9541 11776 31976 583 15233
473: 23303 31003 11553 11554 11555 31546 22414 14396 20378 1420 3488 2382
3244 3833 2429 2255 1958 2842 2428 2151 3480 4099 2856 3227
2056 22825 22826 22823 22493 21906 21905 20582 20580 21904 20583 20581
10877 33242 33240 33241 13085 11556 11557 6036 5691 5587 4779 8393
8110 7180 7150 6910 7151 8648 8112 7133 11551 27168 27167
474: 22618 23303 11553 11554 11555 31546 29205 14396 20378 14949 11107 11106
11105 11103 11104 11109 11108 22826 20580 21905 20582 21906 21904 20583
20581 33246 33244 11275 33245 14105 33247 33243 33240 33242 33241 33239
30394 13085 11556 11557 5222 4322 11551 11552 10481 10483 10480 10482
21094 27167 27168 27169 21913 21914 21911 21909 21912 21908 21907 21910
24594
475: 33449 17641 22413 13717 1206 28799 22039 22040 17684 9785 16937 24638
29235 6275 32114 14316 17263 33329 31635 33036 16027 17892 17893
476: 12205 32928 30726 27835 12542 17994 16100 11182 16155 29585 10900 12578
13423 12676 28852 18351 18370 30692 21038 18558 15710 14603 14498 15186
15026 12910 15126 19020 15532 17506 23779 30961 15515 17779 16246 16267
16298 28092 18793 21190 16674 29863 16842 16883 19236 21883 17179 17462
28540 18742 18685 24734 24758 17972 18110 14027 18490 19365 19309 19303
19842 19609 19762 25972 20994 26797 26665 25649 21707 23204 22328 24311
21011 22246 22250 22841 21292 21227 24175 24181 29965 24000 24654 25864
25860 26584 24280 26974 27689 27792 27752 25325 10722 27109 26441 26326
27301 12046 11514 12370 13343 12534 10399 14941 28159 28178 27365 28388
28126 28492 15363 17996 13656 17995 20260 1826 1572 11566 3559 3470
2359 4034 4077 3232 2505 2582 4096 2123 2360 3439 2275 2229
28984 29253 5243 6220 5735 6623 8229 7152 8004 8023 10575 30684
16305 11234 11161 14754 30889 31248 26873 31420 13163 15394 15475 24596
477: 24861 15227 23325 23313 20850 33492 15069 12210 30227 18531 15339 9952
23316 25064 33627 787 628 33621 567 824 756 613 792 586
720 33628 570 17433 13125 794 32898 31135 14843 14415 17283 10515
31677 15226 10303 30292 16121 26917 17752 29959 16160 31025 17411 12557
31590 33215 24240 25162 21045 13431 12650 12692 18311 11727 13913 18375
14201 21035 18578 14548 17734 14615 14607 17054 14687 14496 15194 15033
12935 32626 15095 19007 15567 17532 22486 23800 15814 23030 15520 17823
28071 16249 28103 18781 21187 16707 26006 16870 32379 27569 16812 20386
29853 15918 11904 23405 23406 21862 21863 20829 17167 17483 17095 18590
21179 26829 18073 28599 18843 18689 16375 24778 18886 19098 465 12832
12052 9894 17962 18130 13987 18495 19406 19310 19506 19661 19746 29921
25964 24467 9702 20335 20981 21005 33175 20547 20528 26634 25638 30133
21725 20806 32206 12344 30336 22319 10089 24312 21014 33367 21590 22260
22861 21422 21309 21238 24195 23241 23135 29483 29960 23928 25721 26056
29990 29998 23166 27513 25088 26586 24262 28278 30027 28844 26981 25207
27661 27656 25326 10707 24983 27114 27059 25288 27159 31044 26420 26323
26197 27969 27256 11360 9527 12045 11474 16482 30624 12367 13350 16220
26156 12506 24585 17923 13275 10408 30519 26015 12252 14921 10827 10595
21006 18247 18248 28162 27345 27349 28379 28146 28449 31588 32872 32873
33180 28669 28668 17680 29309 17437 17224 18373 30013 30015 29291 29290
13659 31636 29453 19733 11816 29695 13265 13264 31578 18649 17603 10474
33620 15923 14277 10539 19995 22212 22211 22210 19194 15382 13494 20148
10347 9575 31803 9574 16625 9971 24627 19515 13000 13001 14795 31974
1727 856 1607 1531 1535 1608 857 1328 871 907 906 905
1403 908 872 1825 1398 1402 1126 1545 1401 1153 1456 1251
1544 1376 1414 1527 1326 1756 1382 1354 1494 1399 1370 1361
1341 1476 1587 1532 1504 1817 1623 1264 1639 1417 1653 1543
31586 31587 33052 10540 23323 23063 4018 1838 3300 3286 3323 4017
3285 2570 2834 2748 3322 4000 2267 2641 3109 3386 3273 2964
9770 2014 2319 2635 3299 2144 2961 2963 2013 2015 2898 10538
2993 1853 2061 2105 9769 3272 2485 2214 2718 2135 2962 4097
2142 2617 2139 2676 2490 2257 2965 2543 2966 2289 2277 3328
4001 2134 2143 1928 2724 1939 2130 2491 2967 1965 2140 2308
2313 2310 2128 2193 1998 4089 1850 2480 2131 1912 2192 1851
2592 2241 2212 4052 1849 2316 2245 1996 4048 2997 2968 2118
3086 2133 1839 2129 2297 2486 2176 4047 1894 2479 2172 2076
2727 1947 4046 2208 4050 3066 2141 1892 1843 1913 2242 2179
1833 1937 2655 4070 2251 2290 9733 32132 23108 20083 18469 33313
14945 10076 19522 28857 32011 32013 32012 16981 28933 29345 29069 29396
20107 27122 11656 20325 23293 13487 23019 14473 9947 9948 19901 13486
21092 15423 18523 20075 20076 33453 33451 33450 33452 33456 33454 33455
32395 23029 31809 23027 23028 19519 19520 32550 19516 32551 19518 32549
14435 19517 23322 23317 29211 10537 21855 10535 15338 23677 23329 23676
23315 5781 4835 6269 6061 5760 5342 4734 4843 4672 18530 18529
5167 5827 6433 5168 4832 5757 5759 5896 5181 6270 4179 4270
4831 4149 4391 6332 4833 4733 6449 6418 4300 4834 5975 4480
5116 4324 4328 4354 5153 6314 6490 4301 4783 6465 4472 4342
4859 10003 4218 4726 4863 4216 4230 4448 4741 4325 4337 4219
4573 4329 4411 4473 4330 4338 4213 4720 4513 4511 5389 5388
5165 4341 4351 4214 4514 5244 4232 5219 4451 4347 5218 5217
5216 4542 4231 4215 4343 4607 4340 5265 5148 4487 4217 4705
4221 5146 5145 4452 5138 4353 5523 4483 4446 4574 5251 5252
5250 5247 5516 4786 4334 4333 4785 6464 6475 5135 10005 4449
4947 6498 4925 4469 4413 4281 4949 5997 4946 4944 4941 4939
4929 4930 4928 4194 4919 4918 4466 5144 32548 10534 23327 6476
4936 4934 4932 5478 4327 5143 4223 4943 4923 4484 23328 4727
4572 4150 5573 4942 4922 4339 4886 4455 4453 5248 4323 6392
4512 4445 5595 4945 4938 4415 4924 4352 4901 6093 23314 33053
33203 4822 5829 5553 6480 5481 4395 10536 4427 5537 4608 4447
10002 31946 23324 23318 23320 23319 23312 23311 23310 9352 7450 7055
7739 8444 7740 9282 9280 8284 6706 18532 7883 15340 8752 9281
7844 7306 8061 8162 8699 8482 8879 8875 8872 8877 8873 8971
8874 8161 8605 8876 8878 8917 8958 8970 6683 7480 7795 6827
8753 6836 8102 7305 7461 7374 7888 7447 8208 7210 8349 8842
7415 8445 7414 8159 7417 8841 8511 7979 7101 8867 8157 6596
7386 6673 15926 9585 16721 9749 20440 17225 20253 13150 16304 10963
707 13608 13609 13626 30452 19745 20679 26828 32546 23004 31283 19524
31589 15337 15336 15335 33630 713 28740 16571 12446 17866 17865 17714
17713 18410 18409 18408 18412 18411 18407 31585 31584 19996 31208 31312
17936 20874 30890 32237 32179 13841 12113 786 32134 22556 32131 23107
32321 32322 23109 32133 32137 32323 32136 32135 21760 21517 23326 16607
12187 26867 11626
478: 22104 32506 33096 10175 23009 23325 23313 33209 27632 32344 32342 30238
30245 30251 9952 23316 25813 25812 17901 787 792 720 628 824
33619 32410 15867 32934 31135 30477 14171 17283 10269 30304 29574 26917
11194 25907 29956 12593 16147 17420 17423 17396 17343 12576 24244 25159
21069 11273 13399 12651 10579 28874 18324 13847 11705 12486 15747 14226
14214 31881 18560 9961 30484 14354 19431 15712 17742 14625 14614 14672
15178 15019 12928 32566 10358 32613 15105 15130 10957 19008 17540 17539
23809 23808 15801 23030 30964 19926 16290 28086 18779 21201 16787 16695
16658 16845 16833 16825 16872 15917 29902 29881 29900 11938 11895 12019
11927 19736 9625 9619 9651 9644 9633 9632 9626 9620 9617 9608
9612 9605 9645 9636 9624 9610 9606 9602 9649 9648 9643 9638
9631 9618 9616 9640 9637 9629 9628 9621 9615 9607 9627 9604
9601 9650 9642 9613 9639 9630 9614 9609 9635 9603 9623 9622
9652 9641 9634 9611 9600 9646 9647 17168 17482 17105 18063 28510
18845 24863 18708 16351 24756 18987 19077 33623 33624 12051 9887 24822
18118 18133 18193 18191 14035 14036 19407 19299 23428 19674 19651 19653
29926 29917 10125 19707 24563 9714 20977 20413 20553 25651 30111 22639
32187 23206 12355 30348 22300 12299 33414 21624 21612 22263 22236 22868
22867 21362 24206 23140 23136 29490 29971 27196 23974 23953 23952 23944
25171 26070 25800 24664 27482 30072 24248 28284 28288 30032 30042 30048
25121 25204 27749 25435 10734 10773 24976 13755 25291 31076 31079 26462
26294 26177 26194 27978 27288 11376 9502 9535 29754 30634 12418 13321
16166 12520 12511 10388 30528 26026 16051 12631 12616 12227 12254 12234
11253 10854 28157 11067 28036 27385 27381 32334 28462 33180 28669 28668
17246 13073 13070 13056 13057 13055 13058 13071 13054 13060 13075 13069
13068 13066 13074 13076 13072 13064 13059 13062 13063 13061 13077 13067
13078 13065 17680 29309 17708 11168 32339 30013 30015 14278 11657 13680
17669 32563 15661 21531 17210 15496 31652 11816 16437 13265 13264 31578
17603 10474 33620 14277 10539 32345 17588 16625 9971 24627 19515 13000
13001 14795 14746 871 907 906 905 1403 908 872 1825 1398
1402 1126 1545 1401 1153 1456 1251 1544 1376 1414 1527 1326
1382 1354 1494 1399 1370 1361 1341 1476 1587 1504 1817 1623
1264 1639 1417 1653 1543 33052 32347 10540 23323 32343 32335 11620
23063 26031 2641 3109 3386 2371 3273 2964 9770 2014 2319 2635
3299 2144 2961 2963 2013 2015 2898 10538 2993 1853 2061 2105
9769 3272 2485 2214 2718 2135 2962 2142 2617 2139 2676 2490
2257 2965 2543 2966 2289 2277 3328 2134 2143 1928 2724 1939
2130 2491 2967 1965 2140 2308 2313 2310 2128 2193 1998 1850
2480 2131 1912 2192 1851 2592 2241 2212 4052 1849 2316 2245
1996 4048 2997 2968 2118 3086 2133 1839 2129 2297 2486 2176
4047 1894 2479 2172 2076 2727 1947 4046 2208 4050 3066 2141
1892 1843 1913 2242 2179 1937 2655 2251 2290 2749 2132 3023
4032 2373 9733 32338 32132 23108 20083 32386 18469 14945 10076 19522
20926 32337 32340 32341 32011 32013 32012 32348 32346 16981 20107 20325
23293 13487 23019 14473 19901 13486 15423 18523 20075 20076 33453 33451
33450 33452 33456 33454 33455 32395 23029 31809 32336 33520 23027 23028
19519 19520 32550 19516 32551 19518 32549 14435 19517 23322 23317 10537
21855 10535 23677 23329 23676 23315 6270 5300 6332 6418 5296 5336
4324 4328 4354 5153 6314 6490 4301 4783 6465 4472 4342 4859
10003 4218 4726 4863 4216 4230 4448 4741 4325 4337 4219 4573
4329 4411 4473 4330 4338 4213 4720 4513 4511 5389 5388 5165
4341 4351 4214 4514 5244 4232 5219 4451 4347 5218 5217 5216
4542 4231 4215 4343 4607 4340 5265 5148 4487 4217 4221 5146
5145 4452 5138 4353 5523 4483 4446 4574 5251 5252 5250 5247
5516 4786 4334 4333 4785 6464 6475 4705 5135 10005 4449 4947
6498 4925 4469 4413 4949 5997 4946 4944 4941 4939 4929 4930
4928 4194 4919 4918 4466 5144 32548 10534 23327 6476 4936 4934
4932 5478 4327 5143 4223 4943 4923 4484 23328 4727 4572 4150
5573 4942 4922 4339 4886 4455 4453 5248 4323 6392 4512 4445
5595 4945 4938 4415 4924 4352 4901 6093 23314 33053 33203 4822
5553 6480 5481 4395 10536 4427 5537 4608 4447 10002 5524 5482
5783 5156 5162 5155 5139 4900 5246 4920 4716 31946 23324 23318
23320 23319 23312 23311 23310 23309 6905 15926 9585 13820 31854 13819
13821 13806 13815 11550 18419 18416 18415 18420 18417 18418 13816 13814
13813 13810 13811 13812 13809 13808 13818 13817 707 30753 22944 32546
16936 13646 30866 31543 15593 33316 11855 33519 31917 22623 33630 713
28751 28749 16509 16545 12455 17866 17865 17714 17713 18410 18409 18408
18412 18411 18407 31239 32567 16719 31189 31228 17287 31324 17709 17936
13785 21701 20861 30910 31996 19913 30462 12139 12128 12127 12124 28246
31533 22993 32134 22556 32131 23107 32321 32322 23109 32133 32137 32323
32136 32135 21771 23326 16593 12170 33622 708 26880 33616 32568 31401
21694 29375
479: 30405 18507 23000 30390 19528 19530 19529 18016 24798 17266 32182 27460
33120 25346 33448 33292 10429 21526 20949 25350 23460 23480 23531 24488
24392 24525 26714 26796 26720 26757 26702 25605 25580 25535 30765 22740
31950 31556 32003 29286 10065 16929 20044 28912 27441 9817 29270 29274
29322 11685 10242 14397 20050 20727 17707 21111 1680 24121 32241 3853
3747 3852 3851 2613 10081 10359 29118 20311 15934 14850 19167 17565
9737 18471 15615 15058 28673 31498 17695 25492 4782 5518 4156 22805
7368 9066 7261 25338 18800 24108 16954 13260 33201 10869 10330 29392
29394 29391 29393 24132 19533 19721 32490 31954 16939 27007 25025 31561
14469 24786 11560 17230 20891 18541 10876 10635 11643 20704 20668 18458
22911 21548 20961 32676 10354 31376 24640 13972 33050 12997 31930 10052
10051 23404 14298 25022 33525 14963 15492 22085 22562 30763 15637 22959
22957 22958 15427 20251 31454 16767 15622 17598
480: 31372 11312 32160 20362 33006 14449 27650 33631 13095 10631 31804 10308
10286 30295 29564 11184 24096 17416 17377 12588 29597 19912 13414 24934
18366 13916 14223 16655 18581 14381 14368 14378 15666 14619 14486 15133
14972 12941 9943 32598 15098 15533 15535 14138 14158 17498 17553 17555
22449 22422 23771 23823 23826 17025 17009 19930 17832 17820 17806 27916
28065 16295 16683 26004 16672 27602 16818 16811 29855 29841 16894 29888
12003 11901 19215 17169 17110 17097 18617 18600 18058 18059 18041 28517
28560 16391 18980 19046 19045 19119 12795 12080 18230 14020 14009 19409
19410 19296 23520 19657 19659 19557 24347 9688 20433 26760 26765 26697
25614 22792 21727 20775 32184 32188 23227 30334 22368 12312 24300 33401
33402 21665 22875 21477 21393 21283 23245 25909 25928 23908 25182 30053
28273 26965 25228 27728 25466 25491 10702 10688 27084 27049 26419 26434
26208 26271 11364 9485 11502 11480 16464 16456 16493 29746 29772 12393
13335 16209 26143 16202 26137 16210 12493 30518 30532 26011 16069 12597
14900 14904 10844 28027 27317 10130 28439 11817 24814 13956 23306 13716
19728 29717 33279 20134 31989 14836 18534 1687 14293 11629 11616 2415
3943 3410 3515 2946 3944 3942 2247 3128 2388 3510 3100 3568
1971 3142 9717 31947 31948 22036 28798 14306 23287 4684 5897 5886
4683 5734 6410 7334 9228 8671 7857 6556 8247 7227 8435 7898
6876 7914 7226 6702 7121 8291 8058 9014 10914 13732 11235 10951
31622 19275 33194 31664 14086 462 28767 31187 31219 31315 31321 31328
31303 20867 20884 30898 24143 12160 12985 12982 12972 14732 21762 26864
26853 15228 31426 13192 13180
481: 33462 12213 27634 27613 30217 760 30222 31539 25803 10349 13101 31505
32924 18668 10345 21672 33598 14953 12537 23707 19274 14517 18628 19997
15594 10241 10339 10340 26609 10495 10310 10275 10306 16104 16128 11175
14422 29947 25898 12568 24838 24243 24227 30005 21075 13409 18317 26882
13228 14212 21039 18556 14374 27120 15717 15698 14577 23896 17059 32887
30191 14504 15196 15035 12909 12875 31377 32601 15531 20106 17529 22492
15084 32417 15794 17028 30978 19928 15523 17836 17795 17828 27894 16294
29821 16291 28078 28226 18792 16789 16791 16790 16679 16798 16799 16779
26001 16863 16823 16899 19266 19248 19225 21889 17182 17088 18067 28603
28549 18847 16369 24766 19106 18149 14033 14029 14021 19336 19814 23590
19489 19596 19683 20483 29925 29913 20017 21927 15746 24996 24498 24390
9678 20983 20425 20563 26653 25607 25878 22779 21720 20787 32195 22355
22303 21806 22856 21304 21225 23260 23257 24070 28261 27417 27190 23958
25170 26057 26078 24653 25867 27510 25097 30026 26960 27673 10744 27110
27055 13759 13771 25277 31047 26185 27272 9538 30632 30602 12384 13330
30510 16070 12228 10816 24104 27984 27377 28385 28140 28455 28428 28325
17248 17574 30767 32130 17432 32128 10529 33495 33561 13651 17626 30766
20948 12488 18504 24025 14447 10205 17580 33475 911 1469 32038 27443
14883 14880 14877 3678 3165 18393 30025 31623 5189 6406 6266 9239
6684 6575 7452 7931 9811 11814 32670 32980 32112 31913 20617 21004
9567 10207 30746 33276 9965 14409 21553 30485 28708 28710 23742 28632
16568 12864 12449 31137 32050 27143 20882 21496 22109 11041 12988 28251
12965 10617 21749 21755 21512 16599 12180 26863 31433 31423 27532 29374
13193 13191 13164
482: 18506 982 1096 3007 2421 2343 28924 31337 6030 5727 4202 6148
5872 5543 5287 5667 6097 5534 6287 5822 4210 6096 9128 8776
9134 8498 8292 6954 9295 7832 8491 9205 7725 9394 8418 32517
33035 15402 17572
483: 20536 32302 17639 17641 23467 23484 24336 24377 24495 26738 26699 26696
26742 25554 25577 25528 22658 22674 22689 22413 1059 22040 22041 22039
16937 17684 9785 24638 23347 23348 15995 9991 6275 5889 8037 9105
16944 14868 11792 10329 13458 18533 20706 14316 33036 16027 14326 11785
33626 33491 14328 14327 25363 21143 11787
484: 23483 23534 24445 26644 26722 26689 25531 25532 22645 18865 1810 1038
1357 1042 1056 1234 1719 1389 948 1108 1690 1185 855 32398
32397 3826 2554 33054 12749 11172 18836 22831 20644 19272 32881 24845
30839 10000
485: 1603 1197 1362 1707 1371 1446 1408 1063 1390 1214 1648 1507
1225 1455 29095 28921 28922 29256 29257 29255 29183 28983 29254 32017
32016 6098 6370 6732 8015 7962 9427 8544 9363 17877 11755 20149
24626 19156 11756 30391 19165 15345 18868 15460 9543 11762
486: 28661 1029 3443 22048 22047 29080 22162 5506 8147 8534 7623 8937
24795
487: 18507 15657 11386 18016 27460 33120 22123 17997 21535 25350 23446 24478
24523 26695 26764 25631 30765 22780 22794 23694 32003 28912 27441 33137
24040 18528 29269 29274 33254 14397 12423 13789 11856 1461 1067 17998
24121 22118 14252 24829 15068 10359 21992 29133 15758 9998 33531 33234
10955 10956 15615 28673 10542 31655 10543 13615 17695 30446 18405 25338
16029 17647 17646 10572 19925 31657 19523 32118 24132 19721 31561 14469
18541 20678 19146 20961 24640 29454 14298 25022 10967 10332 22562 30763
15427
488: 21160 23464 24408 24537 24340 26791 26667 25617 25632 25653 25636 22643
22766 9795 25402 33524 28015 13717 1819 31335 3578 3450 4072 30560
32423 32422 29228 30833 21982 32424 19902 10176 32983 20086 15242 15371
23679 11429 23746 20853 4155 5208 6435 4771 4154 9143 9359 33095
32108 30498 28893 33318 11328 24320 26822 18377 13242 20711 22972 20707
10970 10964 14417 32181 21142
489: 33529 32539 32242 20358 32540 22984 29178 29222 29223 33071 29676 22217
12269 10566 10601 24839 31900 23045 23044 33605 33604 33603 33602 14439
15361 27827 15395 21542 17630 24135 18404 11159 11293 10295 11110 11068
15937 23042 23041 23040 16133 17363 17424 17427 10895 9989 15610 15652
12670 12709 18360 13861 13233 20105 10952 21527 11866 14533 23644 14372
31232 14364 17741 14628 14678 14507 12938 15108 19027 15562 14148 30682
17509 22458 22421 23124 23822 23784 27881 27892 16257 16284 28076 16786
25995 27545 27575 16832 19163 15898 12013 21876 17196 17466 17450 18619
18047 18040 28577 28528 31782 18753 18726 18717 16394 16334 20437 24712
18923 18991 12067 9888 9878 9904 17973 18148 18153 18146 14037 19388
19397 19818 23607 19704 19643 19682 19654 19561 19537 19575 20016 24412
9695 20339 15584 30540 32200 23195 23187 23208 23188 12346 30325 22339
12283 12292 12281 24298 21019 21419 21400 21455 21425 21291 21357 21337
21226 21250 23247 23158 29964 27416 24005 23942 23172 25851 25107 25833
25076 25841 28849 28827 25707 25665 25698 27703 27732 27780 27724 27739
11674 10743 10790 10729 24977 13766 26503 26287 26242 26324 26365 26295
27940 27971 27287 9531 10473 29787 16471 30644 12414 12374 14291 16188
16218 26103 16219 26101 24578 24592 24591 17904 30512 16050 12629 12225
14926 14944 10834 28197 28174 28168 28021 27334 28391 30724 32979 20329
10244 25233 19511 33302 23734 23056 32535 25501 33338 10343 18627 30698
11277 9997 16410 27433 956 997 1570 25521 31936 31999 11577 11621
31541 11622 3647 21669 29292 29403 22139 22144 31616 28975 28974 30691
30690 33212 4392 4965 9191 23762 13449 27520 27521 27518 27519 27517
21926 10514 22922 11148 32542 15373 24784 20633 23049 23047 23046 23048
14872 32571 27144 33213 14826 31800 28724 28695 28701 12452 31223 31173
31182 31300 31307 31318 31314 20868 20880 11858 30942 30931 30900 30937
20725 18099 29213 15929 11017 12123 12100 12979 23052 23051 23050 23053
23043 23054 23055 10556 14167 18470
490: 33084 31504 27620 30254 30272 25065 13122 13113 11310 21670 17692 32585
30311 10274 16101 16129 24081 29939 17338 17307 17332 10889 12577 12570
25157 21073 13394 12647 12690 31284 18323 18362 18559 18563 21779 14527
17070 17050 14482 15160 15211 15163 15004 15052 15008 12894 32300 19033
14131 14133 17550 23816 16999 17794 27903 29825 28091 32376 27539 27551
20500 25976 29894 11910 11955 11919 21866 17113 17134 28553 18676 16326
16315 24725 18910 18896 18908 19135 12802 18217 18177 19346 19833 23547
19601 20013 26682 26681 26683 26672 26676 25559 22734 20595 30368 24313
21649 21655 21604 22291 22871 21452 21361 21246 30180 25177 30095 25087
24252 24272 24250 26949 26532 27089 25307 25295 31041 26411 26222 26304
27246 9511 11500 11501 11499 16477 19905 10417 13316 16205 17919 16059
10596 28029 27335 27350 28420 28461 28498 13708 13654 22062 16612 15624
14085 32511 16411 16434 14097 32977 18259 1034 23332 31805 3756 2285
32076 17861 14070 30712 9735 17651 16632 10884 5614 6144 8612 7474
8613 7476 8614 30718 29742 27524 22942 14871 28744 28680 16547 31170
19735 30948 20588 20613 31446 28049 9674
491: 33464 27846 31629 19719 15168 15012 27922 28083 32355 19198 33211 16383
18900 18111 19300 19349 19293 19334 23505 23496 23564 23614 23491 23591
23470 23455 23495 23488 23560 23620 23636 23517 23523 19587 24337 24513
24452 24528 24334 24454 24375 9684 9693 9710 20426 26774 26643 26704
26786 26773 26813 26639 26652 25557 22705 22681 22767 22758 22700 22723
22738 22763 22652 20804 23984 23994 27504 26485 26247 26288 12267 12240
11251 10850 11245 10825 28012 13702 13711 29729 29680 29735 19469 1135
1523 1137 999 1561 1460 1458 1609 1292 11607 3534 3931 28800
11651 31367 31366 14633 6242 4527 8712 9016 9278 7792 7690 9055
9820 9819 9919 31703 20651 20664 28812 33551 33278 28762 16506 29159
29162 29160 29158 29161 18296 11042 12149 13215 13187 13207 13208 13154
492: 33464 10059 27846 14423 16009 18431 14332 10304 10246 17324 31629 19719
15694 15168 15012 23770 23761 30977 30975 17038 16988 17825 17758 17819
27922 16280 16259 16258 16260 28083 28077 16781 32355 19198 33211 18752
18755 18754 16383 24726 18900 18194 18111 14043 19349 19300 19334 19293
23505 23491 23564 23614 23496 23620 19476 19587 24337 9684 9693 9710
20426 26704 26774 26652 26773 26813 26786 26643 26639 26718 25557 22767
22681 22738 22705 22758 22723 20804 23984 23994 27708 25664 26485 27292
11492 11491 12397 16163 12267 12240 12224 12237 12236 11251 10850 11245
10825 28192 28188 28012 11435 21206 9866 13702 15924 29729 29735 29680
1775 969 1774 19710 29281 26915 19709 11607 2562 3475 3212 26884
14633 8503 8655 6678 10465 9820 9919 31703 29741 29170 10549 10498
18525 20664 20651 13152 21165 28812 33551 18000 33278 16506 29162 29161
29159 29158 29160 11042 12149 12984 12947 13215 28040 13187 13207 13208
13154
493: 23425 24501 22741 29293 1266 21966 4541 5609 4807 5873 5086 5234
4408 20667
494: 23095 23096 25334 25601 32260 32261 32262 1178 1616 1177 1143 1058
1822 1657 1786 1571 1649 1321 1515 3945 2910 3363 4003 1985
3539 14961 22031 29075 32259 4224 5291 4140 5763 6072 5346 5015
5692 5290 8109 8688 7494 8907 9321 6909 6777 8309 8116 8449
8310
495: 875 4082 3603 18851
496: 24860 32169 14729 12206 27621 27630 30252 25805 13128 12217 31794 31795
24032 32905 15745 32724 32720 32725 32723 32722 32718 32717 32719 30447
32721 30448 22511 22510 22509 22507 22508 13252 27829 31793 12543 10516
29419 29420 14741 24830 11237 30312 29361 29358 29357 24684 24683 24682
24681 24680 24679 14728 29354 29363 29362 29359 29356 29364 29360 29355
29365 16106 16127 27859 17753 29943 16159 31026 17402 12551 24223 25138
21066 13430 13371 12673 12711 9805 18312 13874 21040 18549 14528 17053
14688 14514 15180 15021 12898 32287 32625 19032 15548 17531 23799 15787
30543 15516 28070 29822 28085 28105 28213 18782 21200 16865 32377 27588
16813 29843 11994 19252 21880 31769 17184 17461 17102 18591 10796 18069
28614 28525 18842 18690 16374 24777 18966 19041 12831 12056 17963 18190
13993 14032 18491 19405 19342 23418 19499 19662 19750 20479 20453 20019
24524 20342 20978 20569 26777 25604 25597 30139 22628 22650 21710 20785
32205 12466 30371 22381 10092 24306 21013 33412 21599 22235 22860 21375
24196 23150 25916 23929 25720 26060 25791 23165 14322 31792 25857 26596
24263 28279 25785 28842 26931 30140 27652 27693 27758 25487 10672 10774
27113 27100 25289 27156 31039 26417 26213 26180 27970 27255 9526 12037
11475 29784 12390 13349 13297 26108 12507 24588 17908 13272 10392 10404
26022 14920 10590 31785 15071 28161 30741 28003 27369 18007 19474 13451
28129 28450 29455 31373 29422 29421 12595 21102 14323 14325 14324 30744
30743 30483 22554 13698 30742 30730 16448 16447 9670 20194 20264 15450
32668 9815 932 1477 934 933 863 1605 1513 33100 11586 11609
2032 3850 2690 4019 27716 3748 2600 3000 2599 2896 2093 2417
3540 3541 2813 2394 2156 2671 22138 28902 22013 31768 22804 17644
19512 19513 13822 31853 23638 5107 5682 4249 4772 5037 6060 9788
4277 6059 5489 6325 6341 6105 5683 4424 5295 6546 5441 4758
6058 5108 5618 4899 4425 6056 6103 6057 5106 4750 5111 8337
8593 9251 7254 9418 8922 7897 6722 7002 8786 9252 7848 7669
9422 8178 7388 8446 8733 9337 8638 9431 9336 8072 8171 7938
6897 6583 7577 7280 8340 9021 6873 7850 6769 8071 8070 8338
8739 7129 9020 8069 8339 6815 32714 32558 13629 14730 21929 16300
11420 18665 18664 22076 10204 29456 20673 20629 23668 32866 24808 31353
24794 31391 20524 20525 23125 28682 29818 29424 11307 31207 15218 30927
31597 12484 12114 21752 16589 12166 29423 26856 13623 31791
497: 32841 1705 3429 29042 6297 6112 7154 8092
498: 17069 9876 23516 24402 25600 22691 23221 16175 26153 26134 20887 16650
983 867 1023 1660 1739 3963 2879 2059 22163 5786 4621 5952
4582 5775 5193 4396 5399 9199 9257 7806 7454 7572 7462 31131
31132 31130 31129 16646 9447 20640 14395 19271 33183 21588
499: 901 1088 3746 2769 2420 1962 2934 4264 5943 5940 5129 5898
4226 4888 4227 4889 8323 6741 7466 8322 7967 6765 9058 8629
8303 8003
500: 14296 22529 33340 30796 30810 20033 20031 30802 31638 9847 30791 30811
20028 30823 20025 20029 28808 1714 1140 30783 30789 30792 30793 30784
30803 30818 30815 30816 30817 30804 33111 17643 19353 19896 29100 13480
13481 21988 22023 30788 30787 15607 24041 9438 15608 30775 30779 30785
30780 30777 30778 30776 30795 30800 30799 30798 30797 30812 30813 15073
30814 6041 6124 30822 7364 30805 30820 30786 30781 30819 30808 30807
30809 30806 10067 14821 11217 30794 30801 30821 30782 30790 20030 20032
11546
501: 32883 32753 32884 13575 12200 27622 17628 30250 17075 13114 32921 33048
27840 32495 9808 16098 16116 29951 16144 29587 17313 10894 12586 24222
30003 21070 32494 13362 12684 12666 12707 18369 18326 11708 13854 21028
18569 14532 14349 15673 17724 14683 14510 15174 15016 12891 32288 32620
19038 19026 15546 14152 17511 22452 22435 22968 23786 23820 15813 30974
16989 15511 17814 17759 27885 27912 27884 28061 16282 28104 18778 21197
16774 25999 16675 29864 27560 16837 16900 15906 11976 19206 19200 21873
17209 17486 17468 17133 18616 12734 18052 18029 28580 28597 18838 18744
16308 16347 24782 18902 19052 19133 12827 12079 18172 14050 18486 19420
19408 19341 19781 19891 19497 19624 19678 19625 19758 25952 29916 9682
20992 20559 26635 26636 25591 29934 25055 21709 32219 23209 12362 30315
22350 32035 12306 24319 33380 33419 21610 21639 22247 22846 21313 24171
24184 23162 24050 23939 25711 25744 26046 24648 24658 27480 30067 24273
24270 28265 25789 28835 28828 26922 25211 25446 10784 24959 27078 25283
31085 26406 26220 26193 11369 11512 11536 30605 30675 12366 12391 13357
13289 16230 26124 12528 24573 17907 30529 26043 14901 10852 28158 28167
27313 18006 28350 28404 28137 28436 28464 28307 13686 16431 17589 1138
1499 1205 20450 11576 1873 1874 24859 3173 2688 2557 2766 1972
3337 2055 18766 11155 21947 22625 4700 5838 4239 5505 6224 6346
4699 5857 5701 6853 8334 8335 8336 9208 7591 7308 9210 7593
7592 8622 7309 7594 7357 9209 8188 23001 10413 16720 33310 25255
19186 32414 11387 14088 28773 16561 31136 31211 31295 21522 33125 30952
31596 12121 17438 21768 16595 16596 12176 12177 21705 32655 26846 15468
11750
502: 13535 25809 25143 21061 12654 32290 15771 29820 28212 17162 17449 18622
12763 19364 19785 25053 25717 28270 14936 28176 28443 14261 21678 15379
11765 891 1289 892 1904 30590 32009 4246 4698 5784 16542 31146
21498 21487 8627
503: 20534 30289 20537 20535 27455 31523 31529 31524 27454 31531 31530 31527
31522 31525 31520 31526 31528 31521 29416 20533 27876 11193 31092 24231
15352 13416 18319 17060 14503 32619 15080 29849 29837 21881 18609 18730
16330 24688 14873 12077 9902 19400 19338 19790 9677 20995 32404 20551
30131 21722 12304 22228 25723 26058 27488 24285 26967 25688 25276 26179
27299 9462 11494 29782 16485 13336 26119 26118 26154 14903 10815 28171
28384 28457 21104 30736 1294 3722 3153 3721 3136 5288 5272 5273
6117 5493 5271 5274 5656 4521 29815 29418 26826 31164 31595 20839
32952 21765 29417 26877 26875 31434 9017 9056
504: 21132 21133 3744 3208 2664 3595 3921 3753 1956 3225 2915 3444
2264 2984 3664 2236 2665 3130 2248 2746 2183 2747 1924 1878
2204 29132 29202 5547 4951 4952 5324 5325 6195 4164 4165 6417
5630 6436 4676 6345 4802 5564 4673 4416 4356 8948 7597 7080
8771 7219 7081 7218 6733 7013 6663 6996 8266 7078 6915 8244
8140 7079 8468 7465 8560 7765 7077 9069 6670 8141 8683 8715
6756 8013 6845 7421 7004 6927 6842 6993 6858
505: 13574 13142 25144 21081 15811 25054 22274 31088 25408 15407 17624 1392
962 991 3907 3219 31465 22033 6444 4601 6213 4160 6196 4576
4956 21555 16582 21502 21510
506: 896 903 1310 24942 2345 3873 29057 21948 11275 30992 20078 5120
29206 18862 10875 33255 14794 32831
507: 10228 27862 21849 15541 23847 23866 23884 23875 23871 23874 23855 23846
23843 23863 23861 23868 23872 23845 23869 23842 23856 23857 23864 23849
23880 23844 23851 23852 23878 23876 23860 23865 23854 23839 23867 23850
23840 23858 23870 23883 23859 23873 23862 23885 23841 23853 23848 23886
23838 23877 23882 23879 23881 23569 24482 25548 22757 21848 21813 21810
21818 21814 21812 21811 21820 21827 21825 21819 21817 21832 21828 21841
21829 21826 21847 21809 21846 21821 21845 21842 21835 21824 21815 21837
21831 21816 21844 21834 21830 21833 21840 21823 21822 21836 21838 21839
21843 21804 21805 27964 1626 1044 1636 1621 974 1691 1103 1710
976 1781 3825 12721 31951 32015 32031 5099 5752 4407 5611 5028
5082 5093 5021 5098 5097 5398 5096 5245 4818 8227 9284 9248
8782 9283 7534 7711 7860 17042 32707 14254 20663 32711 20609
508: 27616 14831 14224 14344 17737 14580 14675 15113 33132 18501 9908 27131
19796 23526 33136 24033 24332 9699 20430 26712 25613 22761 20807 20764
12349 24072 27500 10720 30164 24965 26325 11377 30607 30501 12251 10842
24137 24136 15307 19730 14763 1437 1186 1304 1369 1788 1166 11406
11606 14964 3929 3930 2536 3147 2537 1918 3117 2535 33258 28998
29400 14250 9950 14855 6038 4819 4768 4431 6001 4298 6152 4299
4381 4443 4462 4794 7056 8772 8926 9274 9273 6792 6589 8773
7518 8297 6570 6614 8860 8861 32252 14388 24908 13461 10966 12445
31293 20855 31276 11547 15940 31697
509: 1231 1576 1293 1314 1176 2106 3881 2920 3882 3029 2954 2053
29074 22026 29067 30660 22020 5693 5560 4331 4830 4383 6371 4504
6201 6166 6523 5396 4517 4960 4860 8910 6557 6571 7176 8200
7513 9202 7339 8199 7448 7205 8089 6802 8783 8784 7764 7477
7026 7027 7221 9361 6744 8350 7829 9426 6870 8678 6879 7934
7204 7608 7935 6746 6705 7997 6971 16639
510: 29944 21068 13879 22476 17029 16995 17776 16702 16850 20490 11962 19238
17205 18084 18958 19063 20469 25965 20987 22710 32185 32221 33392 24049
25873 10673 10384 28152 33216 9721 13676 13677 15411 24639 11593 11592
3759 3760 2755 2333 30862 4823 4398 4297 18436 31389 25499 21499
30923 30924 12958 16610 12189 26833 13194 8363 9387
511: 11124 32402 17602 25406 25407 32527 15149 15148 15037 12933 27889 23503
23524 19663 20357 24520 24464 24508 24554 24459 22695 20772 20771 12286
9533 31992 33049 11650 32004 29168 14072 9996 31991 9999 17260 20127
32660 32426 24630 1014 1277 11300 11299 17077 2859 2858 2024 3141
2659 2661 22055 22012 30836 14752 14631 16011 16012 6291 6076 5085
32381 10328 32382 18872 10527 9750 20656 13232 19899 33528 33564 24128
23899 24129 15592 15639 31142 31639 31640 30495 30494 8057 8056 9400
512: 18586 1522 3105 3676 3677 2312 3106 2065 30213 5484 5495 4606
5485
513: 15400 14267 18660 15431 11745 14266 15453 14269 22966 11786 18661 15466
14829 10222 11746 10221 11744 10220 14268 15414 11768 1366 2510 14750
5856 4310
514: 922 1154 1500 1079 1040 1435 6351 9137 7003 9136
515: 15344 1227 3201 3711 3202 3054 2455 3712 22035 8933 8493 7917
516: 23413 16467 879 2072 3796 3206 3579 3795 31473 29124 6252 5585
6221 5878 9071 7272 9383 6828 8972 7464 9068 7132 7033 7358
7825 6768 7206 8793 8396 7793 7239 8686 7082 7993 7794 7509
8209 7335 6745 20109 11813
517: 30096 15805 23421 23458 24328 24481 26660 25608 22627 24268 31051 1659
1658 1355 3684 2034 2481 2524 30704 21939 23900 24841 22075 15649
5328 4260 5569 6366 5327 4417 8327 6989 8263 8765 8296 8766
8696 9272 7849 8095 8328 18801 23744 32250 16904 20655 23680 28806
32590 15457 9675
518: 21774 33090 27625 30277 25066 32920 13450 33597 27834 16628 16108 16118
24086 29582 17409 12556 30007 13410 12698 18298 13881 11716 26883 14213
18579 14347 15671 14590 14676 15176 15154 15017 14999 12899 32600 15119
14123 22436 15075 15818 27886 16247 16236 28114 16696 27587 15900 29872
12000 17178 17458 17087 18618 18030 18703 16328 18974 19134 12840 12073
9885 18192 19401 19347 23417 19492 19623 24545 9712 20427 20576 26666
25594 25654 22640 21706 20792 20795 32212 12360 30360 12298 24308 33417
21643 22259 22882 21474 21462 21389 21324 21276 21271 24185 23243 24026
24067 24008 30179 26071 27484 26581 24254 30045 26961 25690 25329 10664
27117 31037 26404 26184 27942 11381 11471 30610 12368 13331 26109 26136
12501 30533 26021 12628 12260 14933 11242 10818 28185 27380 28425 29708
14946 11595 7424 8919 15869 23079 20683 24923 28743 12447 31148 31327
20886 30929 21164 12152 21756 16608 12188
519: 24385 22726 22770 1237 1656 1671 1665 926 1410 5512 4616 22943
16537 7023 7143 8484
520: 27434 4035 1963 4421 5094 4168 7955 7763 8647
521: 13522 33039 1189 2561 29090 5130 5793 26995 7195
522: 1193 2230 2231 3391 30199 21981 4656 7167 15422
523: 24343 1346 1099 1693 1117 1540 1772 1375 1203 1204 925 2905
3903 3904 2875 3905 2874 3681 2953 2314 2315 3259 30705 29018
21940 29072 6529 4112 5899 6528 4441 5992 4884 4439 5994 6179
5429 4883 5900 4440 6210 4177 6087 4998 4882 6495 4609 6527
9327 8600 8346 7366 8347 8131 8115 7636 9428 8505 7854 7392
7852 7400 7694 7401 9342 7638 8473 7640 6644 8126 9285 8348
7211 7635 9325 9286 7252 9328 6968 7853 7273 7639 9326 6997
8062 8899 6709 11735 9844 16985 29332 33511
524: 26690 26691 1547 1593 3843 2878 3842 3089 29321 29142 12426 4953
4959 6094 6022 7821 9163 7822 7393 7803 7286 6734 8081 32407
17884
525: 1709 2952 3507 2619 14632 28865 5260 6277 5263 5264 8326 6917
8680 6701 8299 7706 7905 6700 7355 8681
526: 3347 2865 21994 6467 4629 6289 5677 6457 6808 7318 33104 15355
527: 936 1164 1219 1340 1100 1496 1262 1689 28972 20281 28795 7292
9047 9037 6553 17621 17886 16962 11772 31981 33080 11782 24613 33143
24606 33044 33142 15734 19698 19699 15728 33145 33043 33074 14777 24634
33144 17889 32853 16621 24608 32845 33075 28671 33486 15880 33146 24607
20066 16307 11740 17620 31990 32503 24930 11741 11749 19157 33077 29327
22964 15878 15857 18239 17613 20117 33147 33076 33487 32893 33081 31916
11790 33473 14790 11791 33082 32894 32554 15856 589 20110 32520 18238
20118 25309 9563 33078 19700 16637 10223 11757 9552 33073 32829 11751
26823 14273 15391 26918 11747 24603 33102 33083 24617 32275 24609 32851
32836 15860 32276 17612 32834 11748 9540 32651 9542 9556 14280 10046
11752 33483
528: 1413 20299 29830 27422 27421 27435 15469 28786 21854
529: 33141 31510 33527 32507 13564 13577 13548 20224 12198 27614 27623 30281
30270 13138 24124 9793 13735 9666 33447 27832 11323 20190 12422 32069
32067 15294 27875 25903 24083 16141 22135 17403 17408 32079 24221 25156
29152 13403 12652 12693 18332 32070 21023 14546 17739 14593 14484 15006
14998 12920 19009 17541 9792 19772 21693 30957 19923 15619 15509 17757
27891 16241 18795 21182 16671 16657 16856 16810 16814 15895 11995 19196
32066 21868 32064 17158 32068 17444 17100 18612 15085 28598 18848 18719
31545 16361 32065 18967 18973 19055 9790 17971 18109 18142 14007 18485
14232 19391 19875 23563 19496 19493 19693 19638 19614 19751 20480 25973
24475 9703 20334 20336 20970 20574 26658 25624 30134 25035 25883 24126
22796 20802 23178 30344 30340 22308 10083 12295 22277 22878 21467 21377
21310 21268 21239 24204 23254 23270 25927 27199 25174 25175 26074 29983
25871 10800 28837 26955 30141 25260 27802 13021 10724 10776 30156 27160
31087 26433 26345 26198 27929 27968 27290 12039 29767 30642 12420 13325
12508 13269 10380 26010 12600 12022 14908 10826 10598 11285 14886 28186
28470 31801 30849 15258 29714 32072 1791 11567 16726 16943 11126 24122
29283 29314 21990 9990 31924 20592 11141 5326 5732 4847 8981 9246
7324 7796 7675 8643 7418 31374 10892 33553 22207 24323 28231 10361
32073 32071 17573 20223 14438 30683 17256 16530 29813 16556 16570 12435
32997 31179 27431 31956 9959 30925 30899 23395 21022 29973 23286 20610
12133 21754 21494 16606 12186 11441 26878 26858 21209 31436
530: 27129 19701 14100 23726 31747 13792 31746 23725 27866 29279 20965 27582
24694 19796 24441 24383 24437 20430 20428 20429 20406 20393 20408 20407
20421 20411 20389 20418 20412 20416 20419 22659 22693 32186 28254 27174
30060 27670 10669 10720 30161 27252 28405 22901 13657 15307 17078 10530
32998 1111 1686 11147 31371 24816 33205 11603 11606 23016 19527 14870
14425 14869 14424 29267 17943 17890 19521 32025 32019 32026 32027 31380
22560 25504 17978 11262 15240 13280 18392 17751 22124 10098 10097 10095
23394 10099 10096 18379 10101 10100 23393 10102 9662 9663 18386 18390
18389 11094 30865 11439 32999 6407 5689 5853 5852 6478 6258 5353
4280 6416 13976 5665 4136 5215 9976 9975 9110 8890 8098 9349
7514 7300 9348 7299 16959 8368 9347 19713 17756 31555 18457 32891
22952 11329 18381 18380 18382 18383 18387 18384 18388 18385 14094 14093
18391 22894 18864 31733 31728 31726 31725 31724 31731 31732 31727 31729
31723 31722 31721 31720 31730 21558 31687 31685 31690 31684 31686 31717
31689 31688 31716 31719 31718 31715 11024 11045 11043 11046 11047 11015
11044 11036 11026 11034 11048 11027 11035 31259 31275 31274 31276 31272
31254 31245 31277 31250 31273 31278 31263 31244 31260 31262 31258 31270
31266 20051 32677 13256
531: 23522 24386 22651 2518 3485 4067 3159 2126 3103 2283 2681 3590
29145 28897 29064 29065 6226 4666 5892 6214 6377 6066 4102 5114
5498 7539 7720 8541 6558 19894 20642 33148 24631 14272 13973 33149
20920 15864
532: 1041 3868 2943 2763 2045 2757 3325 2377 3256 3097 2937 2591
29251 30202 30201 5626 4646 5307 5802 5306 4258 4205 5027 8564
7122 8083 8146 6825 6677 8734 7379 8781 32856
533: 929 1820 3461 2005 2699 2827 4066 29009 21952 30589 5674 5901
5858 5805 6548 6657 32882
534: 30756 15300 12196 15223 13148 15342 32908 30300 29567 27857 11216 29941
17412 12580 25759 25155 21059 13378 31920 18306 11715 13880 25013 14196
18565 14373 17726 14584 17047 14682 15170 15014 12874 15117 15534 17538
22463 23806 15768 15507 17803 16238 21193 16700 16879 20521 12004 19220
17200 17120 25014 18093 28568 24865 18728 16324 29339 18981 19062 12835
12078 9906 18198 18483 28809 15299 19307 19821 19655 29929 20008 20550
30115 25030 20813 32193 23202 12359 30322 22331 12303 33384 21597 22241
22978 22866 21395 21285 25920 25923 25176 26072 29997 27505 30051 25780
26937 25703 27665 25431 10676 30166 24962 25299 31083 26457 26199 27238
11485 29773 30626 12392 13304 26116 12513 10389 16085 14932 28000 27340
28141 28499 24567 22887 16945 13714 29716 23026 1287 1207 1256 27466
11585 3471 2992 3381 2775 3382 2674 2177 31095 21978 5980 6284
4518 5036 4148 7890 7372 19922 31679 24922 24061 17677 23025 22616
28683 16578 12459 31209 31297 30892 31261 14770 21742 21511 15301 19161
26832 23304 13178
535: 31546 29412 33056 33057 15420 14772 24619 33267 14804 979 24942 22493
29151 21949 21996 31470 15331 33240 11556 20078 6494 5241 5563 4388
7431 7199 29207 11551 27445 27436 10480 10481 18862 24600 9804
536: 30017 30018 27415 33272 17896 22088 20319 20318 30478 23894 32311 32312
14285 14284 29088 29089 30197 18250 14394 17079 33002 24803 6303 7673
3946 3161 2935
537: 31349 13556 17607 15996 23291 33293 9716 33522 30216 14454 14453 19917
28814 10805 31573 11180 25154 13404 22065 15971 22499 15077 17171 18739
18736 19142 18998 18202 20001 23416 23600 23476 19535 24366 24511 24398
24439 24461 24509 18855 18854 26768 26792 26621 26818 26647 26622 26784
26749 25662 25615 25575 25661 25880 22745 22788 26956 10693 27081 27053
23837 11538 12399 28434 28298 20361 14757 20832 31681 20833 28645 30389
13970 28817 28815 28818 28816 28820 28819 28821 30554 30553 9718 16925
16924 16926 32758 18284 19531 33607 11783 20131 11739 15456 21919 10151
20126 21920 10079 20138 30773 19177 10243 15438 21922 32487 11754 21921
19737 15432 13254 31982 20113 9551 29328 32315 16620 11767 20196 980
996 1118 1379 1380 986 1814 1286 1285 1442 1202 1323 1391
1368 1746 31914 19425 18861 20270 29297 17710 29298 26534 19724 31693
18853 18852 14955 22204 3961 3550 3699 2402 3732 2588 3954 3024
2534 3526 3725 31574 2398 2232 3458 2882 3813 3754 3312 3627
3755 3719 3239 3582 3045 3782 4015 2090 3262 4036 3973 3081
32400 3240 22986 18508 22985 20831 32970 9854 14859 14860 22052 30837
20584 29131 28903 28904 28989 29140 29123 28990 29319 28991 28992 28926
32418 29010 22011 29192 21971 21972 29242 30656 21997 28935 21938 15596
15595 20840 29193 25520 29052 30838 29051 31658 31659 32285 32286 23722
31771 31770 14853 28882 32892 17577 21533 15497 21534 17578 23240 23017
15892 23748 24133 17702 23749 32971 14385 23750 32086 28641 22513 31704
28917 28349 17576 16015 9434 33568 13969 29296 13726 21161 32958 32959
6095 6135 5828 4763 5612 6006 6132 5317 6246 4674 6007 5316
4877 4675 6125 4162 5391 5984 5194 5681 5722 5718 5930 5425
5574 4907 5905 4344 4130 6062 4146 6253 4225 7247 7053 7752
7054 20721 8587 15871 8813 9082 8962 9413 9240 9144 8588 7467
6617 7265 7631 7913 6864 7009 8362 7632 7021 18803 7378 6714
8321 18804 8457 8906 8589 8256 9277 8763 8905 6628 8018 6800
7062 7458 6799 8311 6889 8556 9042 7952 7159 9276 7406 8145
6675 9229 20927 8253 8316 8590 7276 8846 8555 6655 8966 6964
14823 6748 9043 8549 9279 7827 6577 8690 8097 8422 7828 28504
19141 19192 33252 30771 20255 14442 31850 22925 32420 32313 30998 31937
27427 24022 30824 20623 15478 19151 20665 20645 28908 22955 13467 13003
22161 21554 28692 10199 20928 24792 31451 32090 32092 32091 9589 29171
10550 29174
538: 29208 13531 30230 30280 25810 13091 32907 27872 29955 17425 30010 13856
14221 14359 17735 14679 15151 14992 12903 15776 27576 29834 9875 18143
19620 24036 24350 25885 22216 12341 21653 22242 21464 21415 21399 21431
21366 21290 21346 21236 21275 21264 23244 30092 25828 28293 25125 25710
27763 27788 10777 31081 26476 26521 26397 26328 26360 11384 29180 11366
9519 16457 26160 26151 30522 26029 26018 26037 12262 14910 28311 11259
13671 33301 25495 1085 10360 9845 6071 6099 8522 8697 15724 7126
8524 8523 8698 15725 14073 11150 10971 31879 31878 16580 12433 31289
20875 30885 26861 31437
539: 865 1050 3620 3849 2046 2972 30829 31476 22006 31477 5961 5654
6965 8716 7197 8027 8028 9235 7034 7900 7443 8974 8305 8730
7036 8428 7208 7035 7290 8653 33490 15383 17881 28332 32521
540: 14846 32317 24858 17213 27635 27644 27615 30253 10565 28915 27132 33465
27133 28813 31678 10276 29563 11187 11197 11189 29588 17366 10225 13360
13421 13381 18309 18328 11706 13848 15628 18574 14383 15682 15693 15493
14601 25493 27458 15204 15185 15043 12923 33314 15116 15129 11644 15079
15788 30967 17766 17796 27893 27917 27921 16276 29827 16292 32362 27557
15916 20487 20506 11952 11934 11916 19199 17170 17481 17094 18056 28522
18673 18734 16318 24721 24763 24722 18937 19048 12768 12785 10462 18183
18199 19378 19304 19829 19869 19846 19830 23578 23465 19608 19647 19538
19641 19588 29914 24380 24449 24353 20572 26662 26638 25596 25606 25560
22682 22701 22772 32226 32220 23229 23199 12300 21607 22233 22271 22220
22248 22265 24216 23151 24055 27183 23950 23967 23930 30175 25749 30176
30187 30076 28831 26920 26972 26939 25225 25221 27795 27794 25318 25317
13027 11673 10731 10691 26531 27076 27099 27088 27045 26502 26379 26307
27934 27243 27269 27244 11374 11371 9537 11535 16487 30636 12410 13341
13307 16228 26094 12503 17920 30500 12639 12226 12265 28182 28184 28196
28415 28416 28446 28467 28448 28308 28304 13653 9722 29718 16443 16444
29719 16445 9773 9667 1674 1187 1396 11569 22152 20300 30463 14554
26542 26540 17716 20956 4631 5551 4547 5606 5725 4183 4815 9050
7453 7289 7140 9254 9146 7946 7360 17044 9848 24031 32537 23003
26603 26543 26539 33471 26538 26536 15980 25017 25018 24789 20697 20713
20671 20677 14165 24825 14570 18657 16517 29796 16562 31308 20854 30847
26999 12132 12966 10620 10610 16603 31399 31405 31430 3667 2362 2932
2017 2294 4002 3245 2390 2923
541: 14847 33198 13532 20846 27626 30225 15868 13147 33574 14643 14650 14649
14648 14647 14646 14661 14659 14660 14642 14664 19428 11417 21673 18297
14838 10252 10325 30299 30305 16125 29569 11188 17345 17375 10881 12584
12575 12574 12562 33214 9868 21180 13437 13377 18310 11712 13868 13885
11717 13855 12854 13947 10646 14220 15463 18568 18550 14520 25199 14581
33022 15132 14971 12924 21583 17242 32629 19000 15545 17497 17504 17494
22423 23777 23773 23825 30986 15612 17769 17857 28056 28113 16699 32367
32364 27567 15894 29908 20514 11948 12016 18440 18442 23412 17441 17153
18042 28600 28579 18751 16332 24727 18934 18920 19137 12813 18200 18175
18164 18145 18208 14011 19402 19835 23714 23525 23554 23548 23479 23419
23573 23461 19593 19556 20010 21915 16627 24345 24521 24544 24339 24420
24372 24484 24426 24534 24388 24536 24460 24548 24382 24564 17988 20565
22981 26767 26808 26805 26750 25564 25598 25562 25625 25589 25626 25886
22661 22660 22784 22685 22697 20810 32215 23228 23183 30352 30365 33431
21618 22568 23010 22275 22293 22270 22876 21270 23656 23977 23945 23941
25872 27502 27489 25079 25831 26594 24260 30030 30152 27711 27730 27778
27815 25458 13032 10680 27082 27067 13763 13748 26513 26399 26466 26446
26472 26204 26354 27226 11379 9457 29764 30677 13356 13303 12522 12526
17921 22546 22547 22545 16079 12632 27127 27423 27995 27330 28392 28447
23088 18519 18515 13662 13719 13689 13652 33182 13488 30749 14639 14644
14645 14667 15259 14446 19771 24813 16407 29727 16398 16420 13620 23021
23022 11818 29265 18992 24125 1520 989 1795 10352 10351 32429 10350
32428 23696 10795 27464 11625 3679 2095 3544 33459 28797 20302 32176
32178 32177 32175 29061 29048 21970 32174 9595 14851 9957 33300 20925
21580 32968 33184 17668 22415 14556 14555 21162 6459 4810 5850 4743
6101 6359 6203 5186 4808 5737 4534 6028 4610 4132 5987 9090
7707 7230 9085 8108 8360 9214 6848 8288 7283 7502 9314 9313
7158 7684 7685 11864 11863 11865 11862 22395 11276 33358 15343 16301
15054 29427 22540 22538 22539 22537 33516 14401 29737 15820 32811 22543
22542 22544 22541 31500 31501 31912 31865 14295 20619 20689 20634 20714
33140 22945 22536 22534 22535 31563 22956 31453 24989 24988 24990 13240
32880 11405 25195 9719 28778 28766 29792 14638 14652 14651 14653 14654
31140 20890 14663 25194 25193 25192 11030 12122 24326 12953 22999 16600
12181 12191 24926 14641 14640 14662 14655 14656 14658 14657 14666 14665
9586 13210 11811
542: 26605 31901 26907 21170 22097 27430 23034 15348 15347 31649 31648 31922
32935 20728 23033 10203 23032 1168 1075 11170 2957 18233 4059 2286
32640 32641 17754 32644 32643 32639 29275 20310 28794 10191 21098 23086
30840 18437 17977 5255 8654 24907 31643 28907 31911 9969 10200 22117
25196 31905 10935 15458 18761 32942
543: 25362 13552 27610 30226 25806 13124 32569 32913 9594 27844 10326 16099
16119 25902 16151 17399 10886 12567 24233 10517 22064 21044 13364 12685
18307 13862 21024 18555 17064 14485 15155 15000 12892 32622 19004 15543
14156 17551 22477 23818 15789 30987 17858 27901 16283 28097 16686 25992
16855 27541 16841 29848 11990 21861 10184 17165 17445 17103 18623 18082
28519 18686 16312 18969 19087 12762 12062 18174 14006 19363 19320 19784
19498 19619 19752 20468 25974 20988 20554 25041 21708 32202 23180 30357
22307 12275 24316 21021 33399 21640 22262 22872 21458 21328 21244 24182
23152 25918 23916 25718 26048 29986 24670 30069 26587 24279 30031 26924
25666 27808 25430 10781 25272 31032 26410 26223 26192 27949 12035 11528
29756 29765 12412 13291 16206 26122 26142 10367 16046 12605 12025 18641
14937 28177 28004 27315 18005 28389 28139 28442 28301 14333 13684 16435
1492 11581 2631 2919 2379 2041 3152 3157 2459 3436 3379 2215
3384 2125 5924 4393 7463 7367 6914 33003 28757 16543 31145 20200
22572 31663 30950 31607 31606 29977 12153 10624 11156 21741 16590 12167
13725 26852 31427 13211 24616 33488
544: 19645 1824 1039 1498 3196 3275 3691 2127 3209 2645 5761 5876
6712 9035 7293 9158 9034 7493 9157
545: 19162 31134 32246 13618 27640 30273 33558 13145 13116 30844 15375 32930
32900 30759 28919 17590 10418 10419 10420 33016 11128 30685 32166 18240
11127 30546 16091 16126 16957 15358 15357 30396 11208 30444 24074 29589
17320 10896 12579 29596 25760 12218 18315 24856 12745 11430 18564 15721
17056 14671 14494 14492 14502 15182 15023 12946 32605 29219 14118 22472
22488 22485 22475 26573 22439 15083 15082 23766 15781 16251 16231 16278
16274 16263 16279 16270 29823 28117 28108 16662 16673 29861 27590 16834
16802 16876 16901 15901 15914 21871 21891 21875 17206 17096 17142 17093
17132 12731 18087 18688 24714 24775 19112 19102 19097 19084 12064 19417
19822 19883 23528 23602 23487 19503 20471 25950 29912 20462 25011 20394
20544 25616 25056 22703 20778 23205 30356 22337 22362 22357 24309 21010
33418 33406 33405 33421 33397 21656 22287 24172 23331 25910 25933 25743
26053 29981 29989 29985 25874 29996 24671 30089 30036 30039 25694 27659
13043 24969 13758 25286 31700 31036 26333 26358 27239 27249 11373 9521
11462 11461 11520 29781 30620 30615 30625 12379 17905 10405 10374 26039
12620 12248 14928 10845 11249 10837 28164 27346 28423 28412 28153 28144
28130 28453 28488 28305 16949 13673 31116 30731 20228 23299 33601 16401
26574 1180 1386 11617 3962 19286 2909 2497 2340 2187 22148 22137
28930 11007 6508 4189 4875 5470 4659 4190 4365 4658 4657 6319
4460 9084 7730 7731 7563 7092 20354 22621 9809 32662 32663 26577
15723 33038 33589 26161 31780 15087 27138 20680 16035 10924 20577 27124
33017 28713 16558 12432 26576 26575 19170 30955 22609 12101 16586 12163
31431 31442 14854
546: 1481 1343 22000 29011 5535 5562 4869 8423 9421 9389 8884 7001
8300 9324 7533 8525 8441 6649 6892 6781 10146 9550
547: 23459 24532 25530 955 910 3854 3654 3529 6108 7343 9102 8038
8402 8834 6801 9101 22101 33510 32633 33283 10045
548: 23427 24436 26641 25620 20132 32653 21210 15232 33472 2058 3632 2411
2033 29189 29059 5070 4287 6518 6413 4915 5047 6183 5071 7703
9123 9097 8040 8957 6662 8041 7460 8546 7940 8938 14960 20643
9653
549: 25570 15753 17623 14774 31107 14575 6600
550: 30664 25358 32575 32576 11458 22100 9442 22916 22915 27850 27841 33457
15059 10287 10259 10247 10299 10270 11195 11185 12724 29591 16148 16137
29590 16146 16145 17372 17304 17314 17311 17413 17407 17380 17394 17297
17390 17357 17405 17349 17318 17301 10906 10880 9818 13386 13385 13382
12699 18334 18325 18348 18357 18335 18352 18358 13917 13886 24114 15353
15354 14519 15719 15679 15704 15674 15701 15709 17728 14578 14600 14493
15159 15202 15188 15171 15003 14986 15028 15015 32293 32303 32306 15110
19014 14136 14124 14119 17524 17522 17528 23781 23793 17010 30960 16990
17033 30958 17017 17039 30963 17036 19936 19934 15616 17777 17760 17843
17771 17818 17856 16268 16293 16253 16296 16269 16288 28109 28220 16768
16670 25994 16664 32380 27561 27553 27562 27566 16804 16817 16805 16829
16839 15910 15913 29438 29437 29441 29440 25977 20499 29873 20501 29904
29875 29883 25982 29903 20496 11991 11909 11958 12001 12005 11912 11940
11886 11970 11939 12018 11981 19214 19241 19201 19211 21870 17191 17202
17474 17479 18601 12742 12736 12739 12729 28570 28572 28531 28566 28609
28596 28573 28616 18698 18702 18713 18723 18745 18672 18694 18712 16393
16387 24739 24700 24695 24705 24703 24748 24757 18986 18911 18988 12778
12764 12765 12822 12834 12774 12814 12781 12770 14019 19385 19380 19850
19801 19806 19804 19857 19848 19861 23570 23589 23475 23497 23434 23443
19488 19628 19564 19636 19552 19635 19555 19577 24562 9696 20417 26785
26800 26633 26801 26680 26674 26673 22684 30316 22352 22311 22351 22358
22333 22377 22354 24301 21636 21668 21606 22261 22855 22854 24213 24174
24214 24209 24212 24215 29478 25930 28258 28260 28257 27418 24001 23981
25722 30177 25191 25188 30189 25715 25714 27511 27512 27478 25853 25835
25104 30087 25077 26598 26942 26941 26940 25223 25673 25684 27706 27682
27766 27748 10756 27108 27119 27063 27056 27037 26479 26474 26449 26408
26437 26456 26447 26248 26366 26234 26318 26339 26317 26251 26296 26389
26266 26218 26355 26252 27279 27266 27253 27283 27281 27214 27277 27289
9495 11533 11527 11478 11498 11515 16458 29747 30599 30672 12417 12373
12371 12372 12385 12407 12364 13310 13309 13351 13308 26133 12516 12517
18824 12266 14943 14907 28207 28163 28201 28203 28014 28398 28354 28400
28372 28353 28369 28472 28463 28489 28473 28494 28466 28320 28302 21101
31699 17872 13694 13693 30653 4336 6172 6134 6328 5874 5228 4636
7426 8149 7442 7573 7006 9115 9114 8988 9207 10917 11272 18809
18832 18833 18831 23657 21175 20638 18455 26881 23350 13805 14246 14283
33001 20435 29515 29504 29517 29449 29448 29516 29447 29503 29518 29450
29520 29514 29522 29519 29451 23038 13645 11854 24123 29506 29556 29512
29529 29429 29511 29432 29558 29434 29430 29433 29553 29552 29550 29431
29551 29527 29436 29435 29446 29439 29442 29509 29557 29444 29443 29559
29555 29554 29445 29513 29539 29534 29535 29536 29538 29537 29531 29532
29540 29533 29530 29521 29507 29541 29545 29523 29549 29543 29546 29547
29526 29524 29505 29542 29525 29528 29548 29544 29510 29561 29562 29560
29508 28689 28705 28761 28704 28714 28770 28679 28756 23743 12865 31167
31160 11859 11032 31267 31247 31249 12146 28244 12948 28252 12955 31532
28245 10615 10618 10609 12169 13218 31408 31402 31416 31409 31404 31429
13158 13155 13206 13182
551: 23506 26685 26687 26795 25611 15467 18759 24624 25365 15408 19357 7559
8416 8728 9168 8844 7884 22209 17983
552: 24486 26748 26746 26747 25599 22722 25379 21677 20227 11810 32830 882
3869 2399 3770 2658 3855 2517 10503 10504 29250 29034 20841 5089
5366 5608 5965 4442 6479 4634 5397 4708 9116 8691 7536 9138
7496 8790 8791 7743 31240 14257 27004 24947
553: 20817 1766 3123 3139 2621 3250 3324 2719 29128 5063 6035 5668
5637 5064 8413 7871 8711 7674 6810
554: 18506 982 1096 1281 1226 3919 3918 3032 3925 3178 28977 29139
30711 31337 5727 6148 6097 6030 4202 5872 9295 7832 8292 8491
9128 8776 7864 6954 6585 9205 7725 9394 7434 6549 32517 33035
33327 33265
555: 30490 9787 24992 13546 31872 32703 32702 32704 31904 27618 32743 32693
32741 32738 32689 32745 32695 32747 32697 11055 14342 14340 30991 29462
29474 29473 29471 29469 29464 29463 29461 29472 29468 29467 29465 29470
29466 21565 21561 21566 21564 21562 21563 21567 17222 633 13102 15761
31493 15947 16973 19951 11292 10253 33349 17330 31287 13877 13929 12852
13945 22533 23036 20213 19777 19775 19778 13005 15706 15198 15201 14990
14987 12919 12924 12914 14798 17503 22453 23776 17770 19950 19943 19941
19949 19948 19945 19942 19946 19944 19947 27918 16777 33613 25983 11972
17159 31491 31492 17119 17155 19971 19973 19962 19970 19969 19964 19967
19966 19965 19961 19968 19974 19963 19972 19952 19956 19955 19953 19954
12726 18051 28569 16384 18906 19111 18213 18231 14013 14047 23714 23502
19779 19604 19649 19681 19670 29928 20011 20009 22976 17869 17868 26770
25660 25049 22729 22373 21598 22296 21392 23160 29477 23917 23987 23992
23955 24642 29994 25815 25109 25839 24259 30155 27697 27767 25473 11663
13042 10675 13738 31059 26427 26263 9493 12523 16075 12259 28187 28032
15366 15362 15364 32729 22522 22515 22516 22517 32736 32734 31683 32877
17946 28331 33498 33502 14271 32827 14071 33530 32828 13661 32393 26162
10161 10162 10155 10156 10157 10159 10160 10158 10163 10154 31751 31750
32876 20185 28330 17954 17950 17951 17956 17953 17949 20158 13015 31753
31752 31748 31749 31755 31754 31756 31766 31765 31763 31764 31757 31758
31762 31761 31760 31759 29268 31494 32513 31490 16427 21001 17234 17239
15949 17948 17955 22090 11832 11831 10477 32957 32956 32875 32874 17952
17947 33503 33504 22098 23911 23907 23905 23906 23913 33500 33499 23904
23902 23901 23903 11833 11835 11834 11841 11839 11842 11845 11846 11848
11847 11840 11836 11851 11843 11838 11844 11849 11850 11837 32878 17945
33501 20159 33497 16931 21576 21574 21575 21211 11449 9853 10441 18651
17659 14445 32460 14443 10440 15451 14444 14805 15406 9441 24636 24595
13736 15731 14270 29333 28860 15474 14281 15434 15730 32844 32502 1787
1767 874 32438 23074 14292 33281 11584 9797 11630 32871 3988 3280
3989 2729 2918 2324 1938 2716 3284 1905 15927 3643 3885 3447
23334 24853 3670 2623 2295 2222 3111 2189 2239 3821 3625 4075
2237 2044 3502 3884 3446 3950 2426 2540 3344 4026 2939 2071
3504 2590 2571 2019 3990 3194 2980 3680 3454 2602 3805 17237
17240 31489 20222 15630 31889 31888 22143 13282 14769 28995 28963 22004
28964 21968 21969 32680 20282 29110 21902 32728 32682 17629 31871 32701
16965 16966 15945 11070 11080 16964 16968 16967 15950 17609 11081 11082
11073 11084 11071 11072 11085 11086 11083 15948 16974 33115 9730 33116
31877 27442 19467 19468 9731 9840 9591 31885 21569 21572 21570 21573
21571 21568 14824 14339 23073 21900 21901 21899 21898 32726 32681 32635
13479 21903 32737 32686 32744 32694 32735 22526 22520 22525 22519 32685
32732 22521 32683 32727 32730 10979 10978 29628 29629 29633 29632 29630
29631 32244 21680 5729 5819 6067 6431 5918 4797 5555 5421 5921
4799 4798 30397 5920 5203 4796 5422 5424 5919 5917 4208 6175
4244 4795 6288 5058 5554 5647 5904 5906 5849 5971 6176 5423
5556 5443 5014 5053 6177 5818 4581 6194 5206 33348 10194 6707
7909 6719 8574 9266 9267 9331 7704 9265 9330 6721 7875 13446
23005 6639 9332 30192 8526 7708 7153 9333 8942 9011 7604 9133
32533 7812 8217 8216 7216 30662 8604 30661 6626 7754 7602 7603
9232 7606 7600 7601 8724 8129 7373 7607 8527 7179 8941 7834
7067 8218 7887 7605 7817 9139 7266 7645 8985 7259 8324 8538
7099 11658 11115 10195 10198 10197 10196 17241 31014 13008 13009 19362
13014 20171 31387 33496 32580 13017 20165 20166 20164 20160 20161 20162
13016 20163 20170 20168 20169 16654 20172 20173 20175 20174 20176 20180
20177 20178 20179 20181 20184 20182 20183 13778 13779 21537 22749 30727
13733 13238 22112 31890 31887 31886 32434 32431 32432 32433 32462 32435
14244 22523 22524 22518 22918 16645 33282 10976 17235 17236 15212 23069
23064 32457 32456 32594 22919 22909 16970 15946 17610 16972 16971 15951
15952 16969 28738 32731 17658 17655 17656 17652 17654 17653 17657 32443
23065 23067 29800 32733 32684 32705 11077 31873 11079 11074 11076 11078
11075 32706 31870 31869 32698 31868 32699 32700 18294 18290 18274 18276
18275 18273 18278 18272 18277 18271 18293 18291 18289 18295 18286 18282
18283 18285 18281 18280 18279 18269 18270 18268 23061 23059 18266 18264
18267 18288 18292 18287 18265 32440 32441 19976 19978 19983 19975 19988
19985 19981 19982 19979 19980 19987 19990 19984 19977 19989 19986 23070
32444 20167 30939 30904 18647 18648 18646 10023 10019 10020 10021 10017
10018 10022 18479 18478 18482 18480 18475 18474 18481 18477 10012 10013
10014 10011 10010 10016 10015 10009 20370 20371 20374 20373 20375 20372
20369 20368 18476 31691 32746 32696 32740 32739 32690 23071 23066 32447
32449 32448 23068 23072 32446 32453 32597 32596 32595 32437 32436 32442
32450 32451 32452 32454 32455 32459 32458 32461 32430 32439 32445 32742
32692 32687 32688 32691 11018 31251 12143 31246 12130 12954 19958 19957
19959 19960 665 13171
556: 13561 10281 14343 15702 12901 30681 17007 17800 28075 27548 16888 19222
28547 24742 18982 18950 18953 18103 17571 19853 23568 23437 19559 19591
25004 24391 25887 22644 12278 21387 24045 25090 10749 13751 26522 26380
26216 27209 16164 30503 27397 27400 27316 28382 27401 11264 30772 27394
27403 30703 27392 27393 17700 10794 31957 20128 14796 33270 1190 1443
14881 13018 27396 3916 2692 2137 28986 4848 6251 4159 6347 8577
8811 7926 32328 31862 10152 20351 20350 27395 32664 19149 20632 33233
27402 23728 23729 31642 18805 28691 29793 20857 16592 18806 10613 10612
27399 27398 27391 26870 27019 13176
557: 27607 13135 32914 16095 29579 17421 12587 13419 12663 12703 12717 13887
20527 18580 14508 15205 15044 32297 14130 22470 22430 19929 17792 16684
16854 27574 29838 15909 29896 20522 11922 12009 19227 21858 17157 17160
17472 17442 17122 18595 18089 18022 28556 15979 18808 16390 31960 24719
24717 18878 18927 19068 19066 12798 12801 12793 17969 18114 19337 19313
19298 19820 19825 19827 19816 23432 19485 25937 25967 24491 24355 24430
24429 20985 26771 22635 21719 20594 22348 22360 12294 24303 33387 33389
21615 21441 21457 21316 21245 29485 27192 25726 29987 24669 25083 24276
26970 27762 25454 10728 27060 25302 31089 26359 27237 27235 27224 9469
11534 13339 13287 26128 10377 30508 16086 14896 14924 28026 28390 28411
28128 13678 16405 1554 1771 1393 1134 1150 1046 1783 1602 1748
1782 1363 1597 1599 1755 1584 1809 1451 1156 1757 1068 931
1806 11591 3282 3071 3148 29098 6037 4111 5323 7696 7093 31692
9782 21202 11874 9776 28684 28759 28775 13799 32389 20940 11224 30946
11876 28248 11230 11231 14421 26847 28053 13165 14835
558: 14764 1045 3022 3605 2422 21933 22134 5536 6460 6544 9107 8888
18862 10875
559: 1381 1030 868 1542 3819 2774 3820 21941 28850 4628 4476 4241
4240 5147 8973 9404 8550 8599 21696
560: 27130 32046 15646 29404 26793 25579 32671 32672 11333 10333 31884 1454
1472 2115 3472 18454 21975 15651 14384 23892 29289 29288 5702 6249
33344 10475 10476 9112 9113 9111 6633 14338 15955 14466 33062 33063
23094 10513 10553 29176 18835 21702 22089 21541 15430 20124
561: 1955 2116 28976 8530 8165 8166 19740 14262
562: 15656 15655 15633 9752 9751 15570 15571 15599 15631 15650 15602 15600
15601 33210 1144 1669 1427 15632 3576 3473 3495 2862 2713 3575
3061 30706 6450 5444 9039 6753 8066 8969 8067 8235 8082 6807
7811 8427 15410
563: 9780 10972 17211 27611 17605 23661 27822 27848 14742 23098 26820 11207
24235 25163 30009 12858 12862 13950 12851 13944 14218 21528 22393 19188
19187 14991 15053 14996 15027 12913 22483 23754 23756 15644 31667 25997
32370 16828 20493 17185 17181 17089 17084 17123 18075 18670 24749 19095
12794 18229 19858 19490 19548 19547 19571 19697 19540 19550 24407 24453
20530 26794 23224 33371 21627 21688 21616 22283 22837 24179 25190 25821
25783 11668 11669 10700 30170 30168 26530 27106 27054 25269 26313 26191
27943 27215 29761 30593 12382 12496 12530 12497 16087 12630 12261 21918
28380 28484 28318 24568 20090 10788 18013 11058 28508 29600 1161 30011
30012 31575 28505 3560 15216 31474 22022 14467 20055 4914 5112 31993
8372 8851 8850 18770 16933 20636 32518 33008 32519 33009 22615 28732
28721 29237 29236 31159 24799 11025 11020 11038 26871 31441 15440
564: 31861 13528 12212 30265 22814 24956 10324 10309 30298 29566 27860 12571
30555 18562 15672 15718 14612 32074 10078 12944 15094 15093 15558 17517
22434 22491 22447 17040 17027 15522 17855 17835 27919 28080 28208 18783
25990 16866 16862 27540 16821 16807 16885 29909 19267 17086 17112 17101
18027 18068 18062 28592 28602 19120 19105 19043 18497 19326 19327 19290
23632 19481 19673 19672 20484 20467 20451 20461 24369 20997 26663 26661
25628 22793 21712 21711 20798 20762 20766 32218 30372 22375 22304 21018
33423 33365 33408 21657 22850 24028 23149 25908 24062 25925 24651 24661
26589 24271 24277 25426 24975 27939 29744 26132 25352 10410 10395 15823
15824 15826 15827 15847 15853 28150 15830 15842 29686 29707 29723 937
1619 2824 3234 2393 10416 22005 31102 15840 15852 32591 32592 12491
15825 15831 15834 33299 15822 15832 15833 15837 15838 15841 15843 15844
15845 15848 15849 15854 32813 15850 15835 15836 6440 7737 15846 15851
15828 15829 15839 9979 24843 20323 18867 18451 10077 24325 24324 28677
28699 28676 15461 15920 15921 22810 14970 22885 12987 22998 21751 26869
14818 28048 10415
565: 18663 27444 23018 15260 15261 32470 33110 9590 11853 12759 12758 12760
22963 24009 9951 20911 15654 20915 20912 20914 20913 19433 19432 19434
13800 31282 13801 13803 13802 22989 22622 1296 32411 32509 11414 11415
11413 11416 11412 2375 14290 29190 14751 31364 31363 31362 31365 11309
20999 21000 21589 10344 13229 23675 5552 8020 8022 7688 8770 33581
21109 17693 10494 10148
566: 2219 1973 3724 20295 4366 5519 6078 6079 9357 7748 8617 6620
7487 8616 9356 7758 8746 8657 7019
567: 23325 23313 9952 23316 794 31135 17283 22061 26917 21041 23030 33625
33180 28669 28668 17680 29309 30013 30015 31392 14451 11816 13265 13264
31578 17603 10474 33620 10561 10560 10559 14277 10539 16625 9971 24627
19515 13000 13001 14795 15304 33261 871 907 906 905 1403 908
872 1825 1398 1402 1126 1545 1401 1153 1456 1251 1544 1376
1414 1527 1326 1382 1354 1494 1399 1370 1361 1341 1476 1587
1504 1817 1623 1264 1639 1417 1653 1543 1651 1590 862 1233
1377 1715 1400 33052 10540 23323 23063 2641 3109 3386 3273 2964
9770 2014 2319 2635 3299 2144 2961 2963 2013 2015 2898 10538
2993 1853 2061 2105 9769 3272 2485 2214 2718 2135 2962 2142
2617 2139 2676 2490 2257 2965 2543 3334 2966 2289 2277 2844
3328 3253 2134 1964 2143 1928 2724 1939 2130 2491 2967 1965
2140 2308 2313 2310 2128 2193 1998 1850 2480 1912 2131 2192
1851 2592 2241 2212 4052 1849 2316 2245 1996 4048 2997 2968
2118 3086 2133 1839 2129 2297 2486 2176 4047 1894 2479 2172
2076 2727 1947 4046 2208 4050 3066 2141 1892 1843 1913 2242
2179 1937 2655 2251 2290 2749 2132 3023 4032 2373 4069 1896
2492 2075 2279 2299 2171 2577 2320 2200 2155 2258 2180 4056
2174 1891 1966 4071 4057 1865 4037 4033 1842 2000 3073 1863
4049 2191 1899 2779 1943 2712 2234 2383 1841 2680 2164 1840
2170 9733 32132 23108 20083 18469 33178 14945 10076 13455 19522 32011
32013 32012 16981 21995 31472 20107 20325 23293 13487 23019 14473 19901
13486 15423 18523 10558 10557 20075 20076 33453 33451 33450 33452 33456
33454 33455 32395 23029 31809 23027 23028 19519 19520 32550 19516 32551
19518 32549 14435 19517 23322 23317 10537 21855 10535 23677 23329 23676
23315 6270 6332 6418 4726 4720 4324 4328 4354 5153 6314 6490
4511 4301 4783 6465 4472 4337 4342 4859 10003 4218 4863 4216
4230 4448 4741 4325 4219 4573 4329 4411 4473 4330 4338 4213
4513 5389 5388 5165 4341 4351 4214 4514 5244 4232 5219 4451
4347 5218 5217 5216 4542 4231 4215 4343 4607 4340 5265 5148
4487 4217 4221 5146 5145 4452 5138 4353 5523 4483 4446 4574
5251 5252 5250 5247 5516 4786 4334 4333 4785 6464 6475 4705
5135 10005 4449 4947 6498 4925 4469 4413 4949 5997 4946 4944
4941 4939 4929 4930 4928 4194 4919 4918 4466 5144 32548 10534
23327 6476 4936 4934 4932 5478 4327 5143 4223 4943 4923 4484
23328 4727 4572 4150 5573 4942 4922 4339 4886 4455 4453 5248
4323 6392 4512 4445 5595 4945 4938 4415 4924 4352 4901 6093
23314 33053 33203 4822 5553 6480 5481 4395 10536 4427 5537 4608
4447 10002 5524 5482 5783 5156 5162 5155 5139 4900 5246 4920
4716 5140 4857 4719 4948 4940 4921 5480 5137 4926 5142 6462
5463 5141 5249 4937 4486 5163 5479 4718 5452 4220 4412 6043
4419 10004 4931 4474 4530 4478 4454 4485 4565 4935 5173 4731
4717 5136 4933 4326 5154 5164 5888 4605 4927 4737 5171 6009
6451 5152 5477 4787 4784 4229 4450 4376 5476 6218 5170 6466
4703 5558 6463 6235 5310 4704 4222 4410 6003 6446 6400 6525
4468 31946 23324 23318 23320 23319 23312 23311 23310 23309 8162 8699
8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 8876 8878
8917 8958 8970 7480 7795 8455 8078 7447 8208 7210 8349 8842
7415 8445 7414 8159 7417 8841 8511 8867 8157 8928 8916 8843
6730 6710 6594 15926 9585 585 32546 23330 23321 21549 17866 17865
17714 17713 18410 18409 18408 18412 18411 18407 17936 21504 32134 22556
32131 23107 32321 32322 23109 32133 32137 32323 32136 32135 23326 33628
568: 10201 19456 17859 14247 11297 14248 11298 32716 11296 14501 23798 15796
15795 10353 19235 19110 19065 19091 29390 23540 23566 23514 23426 23637
23447 23601 23624 23633 23543 23622 23567 23482 23474 20388 26719 33373
26174 25433 18825 18827 18826 11225 29294 29295 14235 1064 1261 1765
14400 28952 31479 11258 30556 17992 17990 17991 17989 15603 15604 5979
6128 6229 6521 5663 5981 6065 4491 7968 8009 6645 8010 8279
6685 8510 33295 17616 10916 18816 18818 18817 18834 14168 14169 24909
10912 14092 11220 20717 16641 10528 29637 11267 29638 11268 31656 24994
17930 13607 17277 31918 23692 10202 31942 28925 12086
569: 1419 3350 4039 2864 3723 3085 31100 20305 6355 6764 7457
570: 23325 23313 9952 23316 814 31135 17283 22061 26917 23030 667 478
33625 712 33180 28669 28668 17680 29309 30013 30015 31392 14451 11816
13265 13264 31578 17603 10474 33620 10561 10560 10559 14277 10539 16625
9971 24627 19515 13000 13001 14795 871 907 906 905 1403 908
872 1825 1398 1402 1126 1545 1401 1153 1456 1251 1544 1376
1414 1527 1326 1382 1354 1494 1399 1370 1361 1341 1476 1587
1504 1817 1623 1264 1639 1417 1653 1543 1651 1590 862 1233
1377 1715 1400 33052 10540 23323 477 23063 2641 3109 3386 3273
2964 9770 2014 2319 2635 3299 2144 2961 2963 2013 2015 2898
10538 2993 1853 2061 2105 9769 3272 2485 2214 2718 2135 2962
2142 2617 2139 2676 2490 2257 2965 2543 2966 2289 2277 3328
2134 2143 1928 2724 1939 2130 2491 2967 1965 2140 2308 2313
2310 2128 2193 1998 1850 2480 2131 1912 2192 1851 2592 2241
2212 4052 1849 2316 2245 1996 4048 2997 2968 2118 3086 2133
1839 2129 2297 2486 2176 4047 1894 2479 2172 2076 2727 1947
4046 2208 4050 3066 2141 1892 1843 1913 2242 2179 1937 2655
2251 2290 2749 2132 3023 4032 2373 4069 1896 2492 2075 2279
2299 2171 2577 2320 2200 2155 2258 2180 4056 2174 1891 1966
4057 4071 1865 4037 4033 1842 2000 3073 1863 4049 2191 1899
2779 1943 2712 2234 2383 1841 2680 2164 1840 2170 4043 2119
3044 9733 32132 23108 20083 18469 33178 14945 10076 13455 19522 32011
32013 32012 16981 20107 20325 23293 13487 23019 14473 19901 13486 15423
18523 10558 10557 20075 20076 33453 33451 33450 33452 33456 33454 33455
32395 23029 31809 23027 23028 19519 19520 32550 19516 32551 19518 32549
14435 19517 23322 23317 10537 21855 10535 23677 23329 23676 23315 6270
6332 6418 4324 4328 4354 5153 6314 6490 4301 4783 6465 4472
4342 4859 10003 4218 4726 4863 4216 4230 4448 4741 4325 4337
4219 4573 4329 4411 4473 4330 4338 4213 4720 4513 4511 5389
5388 5165 4341 4351 4214 4514 5244 4232 5219 4451 4347 5218
5217 5216 4542 4231 4215 4343 4607 4340 5265 5148 4487 4217
4221 5146 5145 4452 5138 4353 5523 4483 4446 4574 5251 5252
5250 5247 5516 4786 4334 4333 4785 6464 6475 4705 5135 10005
4449 4947 6498 4925 4469 4413 4949 5997 4946 4944 4941 4939
4929 4930 4928 4194 4919 4918 4466 5144 32548 10534 23327 6476
4936 4934 4932 5478 4327 5143 4223 4943 4923 4484 23328 4727
4572 4150 5573 4942 4922 4339 4886 4455 4453 5248 4323 6392
4512 4445 5595 4945 4938 4415 4924 4352 4901 6093 23314 33053
33203 4822 5553 6480 5481 5156 4395 10536 4427 5537 4608 4447
10002 5524 5482 5783 5162 5155 5139 4900 5246 4920 4716 5140
4857 4719 4948 4940 4921 5480 5137 4926 5142 6462 5463 5141
5249 4937 5163 5479 4718 5452 4220 4412 6043 4419 10004 4931
4486 4474 4530 4478 4454 4485 4565 4935 5173 4731 4717 5136
4933 4326 5154 5164 5888 4605 4927 4737 5171 6009 6451 5152
5477 4787 4784 4229 4450 4410 4376 5476 5170 6466 6218 4703
5558 6463 6235 5310 4704 4222 6003 6446 6400 6525 4468 4355
4477 4509 31946 23324 23318 23320 23319 23312 23311 23310 23309 8162
8699 8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 8876
8878 8917 8958 8970 7480 7795 7447 8208 7210 8349 8842 7415
8445 7414 8159 7417 8841 8511 8867 8157 8928 8916 8843 6730
6710 6594 15926 9585 585 32546 23330 23321 713 33630 17866 17865
17714 17713 18410 18409 18408 18412 18411 18407 17936 786 32134 22556
32131 23107 32321 32322 23109 32133 32137 32323 32136 32135 23326 571
792 586
571: 14844 33335 17214 22103 21007 32238 17219 17218 30491 32247 20931 9597
13551 32396 27637 33294 27612 27648 30239 30248 30260 11686 11687 18406
33055 32926 33507 25339 33571 18655 14811 27017 10633 32103 32102 15215
13484 11294 10251 10268 10265 10297 11215 11174 29948 29935 24082 24073
29957 12594 17347 17370 17368 17415 17351 17302 17422 17352 17428 17339
17293 17326 17356 17392 17305 17353 10580 10581 9986 25136 13392 31288
18361 18355 18344 18354 24857 11725 13920 12853 13859 13889 12850 13943
13926 13937 11721 11726 13918 13928 13904 13905 11729 13908 13946 14211
31882 24148 20214 19774 19776 13006 10936 15957 15703 15707 15667 17733
14623 14610 14491 14691 14690 15199 15134 14974 14989 14994 12931 12930
12940 12873 12879 28230 15100 15090 15494 15495 15552 14157 17495 17508
22426 22438 22466 22467 22418 22465 23824 23765 23783 23819 15287 17041
17004 30965 19927 17688 17768 17786 17816 27899 27900 27883 28079 16687
16716 16680 16800 16797 16784 16792 16681 16796 16776 16794 16846 32349
32373 32366 667 27543 27546 27564 27598 27592 20497 20515 25987 29878
29876 20495 20507 29907 20517 29905 29870 11984 11889 11967 11983 12014
12010 11980 11936 11986 11891 11943 11998 19264 19247 19230 19244 18441
18443 17149 17138 17106 18045 18032 18037 28512 28551 28515 28548 28558
28513 15238 16386 16377 16367 16385 16335 16321 24736 24752 24724 24691
24729 18932 18888 18877 18942 18893 18914 18883 18881 18949 18933 19131
19071 19075 19100 12824 12796 712 33624 12054 18167 18214 18169 18152
18188 18113 18203 18147 14004 14012 14022 13989 14040 14048 19368 19845
19859 19793 19837 23713 23629 23586 23575 23451 23452 23582 23492 23596
23599 23469 23499 23527 23572 19595 19680 19597 19675 19562 19585 19648
19690 19543 19586 19669 19687 19602 19991 25941 25966 25000 24381 24378
24371 24394 24470 24389 24516 24358 24370 24527 24356 24471 24487 24560
24397 24466 9694 20348 17979 22982 26706 26707 26629 26650 26782 20937
25586 25588 25646 25610 30752 30113 23737 22686 22724 22709 22711 20801
20593 20808 12474 12467 12480 23192 12357 30354 22365 22321 22299 22372
22320 22345 24297 33391 33390 21608 21623 21594 21664 22245 22289 22877
22851 21411 21439 21444 21428 21418 21405 21438 21454 21420 21345 21321
21293 21314 21341 21287 21356 21398 21307 21265 21260 21230 21257 21282
21217 21223 23253 23256 23258 23268 23137 23146 23128 23144 24048 24046
29480 24051 25931 29972 27189 23957 23948 23998 23973 23993 23988 23968
23918 23965 25742 25183 25189 30188 30184 26063 27477 25092 30081 25818
30058 25078 30084 30066 30057 25823 25101 30086 25075 25094 25081 25080
25846 25843 25820 25832 30068 25847 25850 30064 30046 26947 25683 30145
25219 25676 25213 25668 25205 27683 27672 27725 27800 27797 27746 27805
27718 27790 27818 27787 27775 27799 27786 27750 27816 27774 25322 25453
25464 25310 25311 25429 25471 25441 25468 25475 25457 25332 13044 13023
13033 13037 13025 13030 11679 13029 13040 13039 11681 11666 11671 10779
10678 10697 10755 10682 10745 10739 10747 10765 10696 10737 10761 10764
10714 10767 27073 27043 13756 13775 26448 26501 26509 26431 26517 26488
26464 26507 26421 26468 26467 26401 26445 26428 26519 26425 26282 26253
26321 26372 26300 26257 26255 26243 26269 26387 26228 26273 26310 26341
26342 26301 26291 26238 26352 26393 26381 26377 26274 26272 26337 26303
26299 26340 26332 26297 26239 26314 26268 26375 26277 26364 26384 27975
27261 27284 27297 27227 26396 9453 9458 9463 9522 9475 9487 9492
11542 30598 30649 30680 30646 30639 30619 30631 30648 13314 26107 16223
16212 16208 16216 10400 10401 16049 16065 16066 26033 26013 16063 26023
12603 10973 12220 12245 10823 10840 27018 32757 17860 27992 28010 27999
27988 28035 27985 27998 28030 27383 27322 27363 27321 27323 27357 27324
17927 17928 28368 28364 30723 16037 28497 28427 28482 28294 28316 31502
32384 27202 16950 14969 23075 17247 10357 20088 17591 15397 19159 15367
18371 18372 26887 15744 15760 22183 14391 20837 10647 23238 11410 27165
27440 17074 13701 13718 13687 13648 13703 33357 23721 20935 9740 22202
29186 16399 16451 16438 29681 33359 29690 16442 29724 29683 13845 17229
14109 13611 30557 23665 14852 13520 27457 21559 14884 11638 11579 9754
11613 11619 33614 11612 11563 2027 2249 3844 2030 2682 3700 3987
2773 3704 2949 3835 3761 2084 2381 4005 2683 3767 2633 3895
2894 3229 2737 3087 1910 2634 3336 13610 11659 22150 22140 22154
22155 22156 22046 29033 22045 29129 28978 21850 31949 11639 11436 33317
29341 6212 4971 4362 6137 5645 5284 5149 5746 5061 4503 5230
5232 4575 6136 4639 14564 7316 7627 14563 14562 14561 7481 9355
7865 7761 9038 9127 9294 7325 7185 28780 8831 7655 9222 8000
6760 9184 8429 7759 7478 8672 7550 9401 9221 9178 8669 7437
9179 7783 7394 7802 10856 21538 21539 21540 17043 32329 20268 31867
32476 33610 32117 21481 10608 24828 25380 14260 20620 20647 20669 20699
25024 31394 31393 21208 33304 26886 31866 17873 13239 11304 30694 14303
17568 15876 9596 23829 28697 28722 28696 28742 28754 28753 28768 12996
11809 16553 12441 12462 12438 12437 24921 31141 31163 31181 31174 31151
17902 30917 30897 30928 30902 30936 30916 30903 29212 24146 22203 17755
24939 11145 9851 9852 11037 12138 12094 12090 12104 12952 12961 626
28247 10605 22997 32972 24933 31445 31422 31439 32037 28047 31553 13197
13189 13169 13185
572: 17934 10050 18253 19351 24422 10787 26717 25527 22692 22222 26595 25445
10759 30020 25012 25257 25256 18439 19727 28637 28636 27013 31633 32995
24946 11265 15959 1578 9786 32755 32756 32390 25518 25519 13052 13053
13051 11608 32240 17278 20436 18510 17747 13483 32075 28822 29225 28858
22830 17660 30376 21963 28962 21962 21961 29043 13727 32824 29389 10071
10072 13482 13959 33552 29641 30455 32994 20266 14299 5199 4274 5970
6317 6442 5926 4166 6323 4371 4169 5890 5198 5490 4759 4745
4744 5969 6441 6223 7816 7446 19702 8295 7630 8714 7815 7240
8080 6890 8521 33094 15973 31359 31360 31358 22213 25360 32124 11359
32045 18398 18395 32254 30826 16976 10245 32049 20843 30550 27000 14809
573: 14234 11052 19938 29860 29862 12007 17082 14018 32199 28276 25670 16491
16190 21560 31986 31983 1244 4009 3818 3186 4007 2971 2622 2109
2969 2970 4008 2579 3187 3817 2764 3267 32500 13617 5895 5055
5176 4515 5999 4131 7338 7106 9304 8731 6809 7522 8087 7337
8088 6679 6902 7659 6618 8417 10584 10585 9822
574: 13007 25538 30017 30018 27415 1628 1448 30478 19999 14285 20934 24039
14263 19893 18250 14394 17079 33002 13728 24803 24804 24805 6830 14965
14966
575: 17214 30491 17219 22103 14844 27438 33461 32238 21007 17218 9597 32396
27609 27612 28241 30283 30221 11686 11687 33055 32903 32933 32926 33507
25339 10497 14414 33571 22612 18655 15067 32103 32102 15215 13484 11294
10262 10266 10297 10251 10265 10263 10268 11174 11215 29957 29935 29948
17410 17353 17415 17347 17293 17368 17339 17428 17326 17392 17302 17356
17370 17422 17305 17352 17360 10899 10580 9986 10581 24224 25136 13392
13373 12661 12701 31288 18361 18355 18344 18354 24857 13859 11729 13908
11725 13920 13924 13889 11726 13918 13937 11721 12850 13943 13928 12853
13926 14211 19774 13006 19776 10936 15957 14538 23643 15703 15707 17733
21093 14680 33020 14491 14691 14690 15206 15199 15138 15134 15045 14989
14980 14994 14974 12879 12878 12915 12931 12873 12917 12930 32301 28230
32610 32627 15494 15495 14157 17495 17496 17508 22443 22426 22467 22438
22418 22466 22465 23819 15287 23765 23767 23783 17005 17004 17041 30965
19927 17688 17782 17785 17816 17768 17786 17789 27900 27883 28079 16681
30449 16794 16687 16784 16796 16716 16797 16685 16792 16680 16846 32351
32366 32349 32373 27546 27564 27592 667 27543 16873 15903 29890 29878
20497 20517 29870 25987 29907 29876 20515 20507 20495 11908 12014 11891
11984 11986 11998 11967 11983 12010 11889 11936 11980 19234 19244 19230
19264 17183 17464 17144 17149 17106 17244 12728 18035 18045 18037 18032
28513 28610 28551 28558 28512 28548 28515 24752 24724 24691 24736 24771
24729 24707 24732 18975 18888 18883 18942 18933 18949 18877 18893 18914
19131 19139 19071 12767 12054 18167 18203 18214 18113 18147 18152 18188
18169 14038 14025 14004 14040 13989 14022 19368 19329 19859 19793 19845
19809 19837 19841 23713 20104 23629 23586 23452 23499 23575 23451 23537
23599 23457 23582 19480 19487 19675 19585 19602 19595 19562 19669 19680
19543 19597 19685 19687 19648 19690 19586 19991 25949 25941 25966 25003
25000 24370 24470 24371 24381 24378 24516 24394 24389 24356 24466 24560
24527 24358 17979 26629 26706 26707 26650 25586 25588 25645 25646 30752
23737 22686 22724 22709 20801 20593 20791 12474 12467 12480 23235 30354
22332 22365 22321 22320 22299 22372 24297 33391 21614 21664 21623 21629
21608 22877 21436 21420 21454 21411 21428 21444 21418 21404 21391 21353
21307 21356 21341 21287 21321 21345 21398 21293 21300 21221 21272 21217
21257 21282 21265 21223 21260 21230 23253 23258 23256 23268 23141 23144
23146 23128 24051 24046 24048 24053 29480 27189 23973 23957 23948 23993
23968 23998 23965 26063 30074 30064 30081 25847 30066 25850 25101 25846
25818 25092 30068 25080 25820 30082 25075 30057 25834 30058 25078 25823
26580 24247 30046 26947 26933 25218 27701 27672 27683 27800 27786 27775
27725 27726 27807 27746 27805 27718 27816 27797 27774 27771 27750 25452
25441 25332 25453 25464 25461 25471 25322 25457 25311 25310 25463 25429
11681 13044 13023 11671 13033 13037 11679 10681 10737 10697 10682 10761
10779 10767 10739 10696 10747 10755 10752 10678 10745 27073 27097 27043
27035 13741 13740 26443 26448 26501 26467 26509 26421 26468 26507 26464
26517 26440 26488 26243 26236 26321 26352 26255 26228 26301 26314 26372
26239 26282 26341 26310 26272 26253 26300 26273 26257 26303 26269 26375
26274 26377 26268 26332 26337 26393 26291 26207 26387 26182 27975 27284
27297 27261 27227 27218 26396 9453 9458 9496 9522 9475 9500 9492
9476 9463 9487 11542 13314 13299 16198 26107 16223 24582 17917 10376
10400 10401 16049 16053 26033 26023 16065 16063 16066 26013 12603 10973
12245 12220 32757 27998 27992 28007 28035 27357 27314 27383 28368 28367
28364 28357 30723 28427 28482 28497 28294 28316 32940 14969 16950 10357
11544 17591 19159 15397 18371 18372 26887 15744 15760 27440 33185 17074
23238 10647 13721 13687 13701 13648 13707 13703 24916 9740 22202 29186
29701 16399 29724 29683 16442 29681 33359 29696 13845 17229 14109 10341
13611 30557 23665 17585 14852 13520 27457 24139 21559 1149 14884 14878
11572 11619 11638 11579 9754 11612 11563 13796 2249 3844 2773 3761
2084 3835 2949 3987 3704 2381 3700 3895 2737 2027 2030 2682
2894 3229 2683 1910 2633 3926 2800 3991 14452 11659 13610 24827
22150 22156 22046 22045 29129 29033 28978 19355 29130 21850 31949 11639
11436 33317 14568 5284 6212 5061 6137 4971 4362 5645 5149 5230
5746 4503 5232 4639 4575 14561 7481 9355 14564 7316 14562 7865
7627 9038 7761 14563 9294 7325 8000 8672 9222 8429 7759 28780
8831 9127 6760 9401 9184 7185 7655 7478 7783 9221 9178 8669
9179 7550 11132 21538 21540 21539 32329 17043 31867 20268 32476 33610
32117 10608 21481 25380 14260 20620 20669 20685 25024 31394 31393 21208
33304 26886 31866 13239 11304 17568 30694 14303 15876 23035 19280 9596
26994 14737 11815 23829 28696 28697 28726 28722 28753 28719 28335 28754
28768 20578 28742 12437 12462 24921 31165 31204 31155 14091 17902 18658
30936 30884 30902 30903 30916 30938 30928 29212 24146 22203 24939 11145
9851 9852 11037 12138 12090 12094 12960 626 12952 28247 12961 12963
10607 10605 22997 22832 16585 12162 26879 31422 31439 31445 32037 28047
31553 13185 13169 13197
576: 32547 31133 13543 12207 27641 30269 13140 32897 30760 33013 32501 12536
32421 11232 27424 20146 14735 23709 10278 30293 29571 27868 25895 29958
14856 17388 17323 12558 30006 15249 15252 15247 15250 15245 15246 15254
15257 15256 12659 13915 13734 14193 21026 18577 14535 14363 17727 14620
17065 14477 15193 15032 12902 32304 32606 15099 19029 14125 14134 17537
22448 22461 22428 23805 15283 31093 15775 30968 30969 15517 27879 16242
18780 21192 16709 29832 16860 27552 27565 16820 29844 16895 15904 29898
25988 11924 11987 19226 19224 23639 23351 21864 17198 28348 17465 17127
16946 15253 15248 18060 18020 18094 28554 28567 18844 24745 33625 18963
19044 19082 19127 17966 18225 13985 18492 19325 19855 32107 19505 19658
19589 19761 20478 20456 20337 20974 20410 11881 26701 30121 20803 23233
12335 30327 22349 22336 10087 12277 24317 33368 33396 33427 21591 22280
22253 22865 21379 21277 23145 29970 23932 26081 24647 25870 23171 25085
25089 24274 25124 28829 25699 27699 27660 27654 27655 27653 27768 25481
10711 24986 27103 13772 25263 25271 27154 26322 27931 27976 27212 29233
11363 12038 11487 11472 16494 16453 12394 16196 26110 26130 16168 12515
24577 17903 13277 10378 30526 18821 18822 26030 16074 12622 12257 10593
28019 27348 15251 28421 33162 24846 13660 30745 30737 15255 16426 16417
29731 29734 29713 29679 16423 23897 17581 9546 32852 33319 20153 15436
31953 32825 32826 17599 15635 31973 1812 1020 1215 1720 1577 953
1220 1546 1316 1339 23298 33512 11628 25388 2213 15763 15762 1933
2652 3845 31666 1868 3221 2743 2744 3658 2742 3555 3897 3807
3682 3815 2793 2772 2503 2418 3075 2530 3601 2792 3432 1935
1980 2529 3814 2843 1898 3361 1890 3203 2752 2723 1867 2794
3740 3375 2450 1934 2378 3683 2203 2578 2293 2339 3762 2223
2642 3863 3739 2184 17289 32984 29056 31096 21979 29091 29310 30713
32007 14402 24850 17667 6013 4585 5616 4596 4586 5958 4967 4996
4197 4191 6492 4994 4578 5487 6522 4142 5126 4681 6499 6389
5604 5963 4963 4966 4680 4904 5275 5588 4620 4995 5262 5706
4993 4402 4308 4964 6348 5277 4151 4152 4775 4317 6004 4522
6504 8100 8175 9220 27450 9345 8307 9303 9188 6751 7749 7012
8184 7103 7575 8805 6708 8333 7576 7382 6597 7654 8176 8238
8182 8181 9302 8180 7356 9036 8543 7011 8754 6844 6851 8179
8999 8709 8708 8710 8205 8250 7225 7651 7404 6691 6908 7196
7524 6835 7652 9048 7483 9301 7165 7649 6798 6658 7971 7383
8118 32867 9983 10049 18810 18811 18812 24102 11165 18828 21536 21203
33556 10499 33555 13658 14087 11794 16513 16982 31301 20865 30891 13840
12117 12968 10611 21490 26866 26857 13217 32493 32523
577: 9570 9571 1348 1188 2549 1961 2358 1887 2476 3339 3383 2753
4040 2754 3129 2202 3080 3377 2566 2648 2329 2087 2157 2771
1959 2625 1844 2563 3351 2928 17286 29113 29112 14749 14748 10177
14090 6367 5699 5103 4311 7616 8569 8442
578: 33046 22935 10215 10214 31713 31712 10213 10212 31714 23445 23486 24489
26723 26703 25584 25550 22773 22764 31786 11130 9443 10427 10948 28339
28338 32385 1574 1440 2001 2311 2423 3967 26827 20059 30544 10131
29001 29350 18874 32469 12998 19708 10953 29231 29232 4688 5724 5866
4686 4687 5056 5865 6365 5134 5161 4528 6419 4566 4840 7587
8620 8799 7595 8800 8996 9249 8472 8764 8272 7596 8228 16956
30456 17076 33133 10479 14569 9926 13231 22973 20659 9924 27461 29154
29157 29156 20611
579: 10652 26605 29336 29337 24953 26604 31901 15972 22181 31559 21168 21170
21169 13227 22097 27430 32943 31557 13971 30538 30539 23034 17894 15348
15347 33594 31649 31648 32094 32093 31922 32935 31558 20728 20079 10203
898 1051 1884 32640 31940 17754 32644 32643 15931 15930 22550 29395
20292 32022 10191 21098 23031 26524 23086 13495 31816 31645 31644 30840
18437 13974 17977 20103 28642 31650 4909 4761 23355 6376 5360 6412
4689 26902 26903 26904 5947 5362 6310 4682 7837 8282 9160 8837
9219 7322 8838 6958 13782 13781 13286 26993 16652 28907 31911 14189
9969 22117 15981 25196 31905 10932 10931 10934 10933 10930 31802
580: 18507 15657 11386 18016 27460 22123 21535 17997 25350 23446 24523 24478
26695 25631 22794 22780 23694 32003 29286 28912 27441 33137 24040 9817
18528 29274 33254 12423 13789 1067 1461 17998 24121 3671 3107 2145
3108 3614 2233 3341 14089 14252 24829 15068 21992 29133 15758 14850
9998 11736 15615 28673 13615 17695 30446 4957 7149 6938 8621 7813
16029 17647 17646 10572 31657 19925 19523 32118 24132 19721 19533 32490
31561 25025 14469 17230 18541 19146 20678 20961 29454 10967 30762 30761
30763 15427 14634
581: 24077 14353 17049 24733 18952 18227 12301 25106 25845 27734 25296 26261
26369 1916 2866 2404 28875 29093 6191 5333 6301 5605 5332 4642
6278 4536 5009 6081 5334 7753 8929 31326 20863
582: 17523 23792 15280 27920 20509 11941 23440 23450 23561 23555 23558 19603
24404 24526 24480 24561 24411 24506 31007 26751 26737 26721 22797 22713
22252 23925 23956 10775 27306 9528 1229 1412 1151 1423 1563 889
1521 1450 11565 2808 20314 11457 11138 5779 6536 5329 6050 5773
6211 6375 5834 4532 4481 4372 4385 6286 5331 5548 5330 4580
4599 8496 9568 31808 21679 10362 20723 27205 20929 28731 12109 17619
13523 30496 16643 15236 24593 15736 11758 20135 16647 15386 11766 11759
30497 11763 32392
583: 14764 22414 29412 1730 24942 3910 1922 2632 2555 3613 2899 2556
3258 29016 29104 29000 20294 29200 29252 29134 26906 11275 33240 33242
30394 20078 20254 15374 5990 5907 5803 7128 6994 9405 7927 8911
9397 29207 10480 18862 21911 21913 24600 9804 9541 11776 472 31976
584: 21676 14951 30234 25811 13087 30843 17261 22555 25158 21067 13420 12672
18979 17730 14627 15106 17535 15807 27908 16254 16659 17189 23121 33045
18715 16357 24715 12804 9886 19394 19823 19502 19582 29932 20422 30122
12340 22240 22863 29963 27182 23971 25072 28264 26971 24970 31080 27233
29785 12400 13340 28190 28413 9560 15378 4064 3556 2282 3396 3255
2361 18255 5826 4885 6180 16567 31184 12137 11660 31388 11305
585: 32754 13529 23325 23313 17648 20851 12214 27639 18799 23335 30235 9952
23316 30098 33627 33621 824 756 720 586 787 792 628 613
33628 570 567 13117 33619 814 794 815 759 32922 31135 17283
12538 22061 23277 16092 16122 26917 24089 16161 17300 12547 24229 30004
21063 13417 13435 13376 13375 13436 13383 12679 12675 12715 18321 13882
21029 18545 15716 17061 14497 15141 14983 12867 32608 19005 15554 14146
17533 23801 15817 23030 17030 15524 17852 28067 16235 18785 21198 16692
16656 16864 27536 478 667 16815 16887 15919 11906 19231 19253 18625
21885 17166 17484 17083 18603 18071 28605 18841 18709 16349 18890 33625
19103 12791 33624 465 33618 33615 712 33623 12069 18201 18140 14000
18498 19416 19345 19863 23515 19507 19677 19756 25946 29911 25958 25957
24376 9707 20343 20998 20570 26787 25639 30138 22669 21723 20765 32208
23234 12347 30328 22297 12314 24318 21020 33410 21630 22282 22862 21390
24205 23159 25917 27197 23927 30178 26090 29991 30000 27491 25071 26593
24282 30047 28830 28848 26968 26985 26936 26935 26986 25687 27663 27657
27803 25315 11664 10725 10772 24974 27096 27066 27051 27052 25275 27162
31055 26455 26217 26196 27960 27294 9503 12030 11504 16469 12409 13337
13354 13302 13355 13301 16201 26138 16167 12505 24579 24580 17906 13278
10403 10391 16076 14914 14894 10821 10599 28165 28023 27344 27352 18012
28151 28459 33180 28669 28668 17680 29309 30013 30015 13712 17257 31392
14451 11816 16440 13265 13264 31578 20889 17603 10474 33620 10561 10560
10559 14277 10539 17587 33634 14789 15428 31851 16625 9971 24627 19515
13000 13001 14795 1008 1145 871 907 906 905 1403 908 872
1825 1398 1402 1126 1545 1401 1153 1456 1251 1544 1376 1414
1527 1326 1697 1382 1354 1494 1399 1370 1361 1341 1476 1587
1504 1817 1623 1264 1639 1417 1653 1543 1651 1590 1642 862
1233 1377 1715 1400 33052 17251 10540 23323 11597 477 23063 3332
3953 2689 3952 2736 3832 2735 2698 2641 3356 3109 4093 3333
3386 3273 2964 9770 2014 2319 2635 3299 2144 2961 2963 2013
2015 2898 10538 2993 1853 2061 2105 2269 9769 3272 2485 3530
2214 2718 2135 2962 2142 2617 2139 2812 2676 2490 2257 2965
2543 2966 2289 2277 3328 2134 2143 1928 2724 1939 2130 2491
2967 1965 2140 2308 2313 2310 2128 2193 1998 1850 2480 2131
1912 2192 1851 2592 2241 2212 4052 1849 2316 2245 1996 4048
2997 2968 2118 3086 2133 1839 2129 2297 1894 2486 2176 4047
2479 2172 2076 2727 1947 4046 2208 4050 3066 2141 1892 1843
1913 2242 2179 1937 2655 2251 2290 2749 2132 3023 4032 2373
4069 1896 2492 2075 2279 2299 2171 2577 2320 2200 2304 2155
2258 2180 4056 2174 1891 1966 4057 4071 1865 4037 4033 1842
2000 3073 1863 4049 2191 1899 2779 1943 2712 2234 2383 9733
32132 23108 20083 18469 14806 14945 10076 13455 19522 32011 32013 32012
16981 29015 20107 27123 33311 20325 23293 13487 23019 14473 19901 13486
28336 15423 18523 10558 20075 20076 33453 33451 33450 33452 33456 33454
33455 32395 23029 31809 23027 23028 19519 19520 32550 19516 32551 19518
32549 14435 19517 23322 23317 23727 31702 10537 21855 10535 23677 23329
23676 23315 5935 5934 4350 5475 5473 5131 6257 4828 5471 6394
4669 5711 4827 6270 5132 4663 5474 6332 5472 4668 6418 4354
4324 4328 5153 6314 6490 4301 4783 6465 4472 4342 4859 10003
4218 4726 4863 4216 4230 4448 4741 4325 4337 4219 4573 4329
4411 4473 4330 4338 4213 4720 4513 4511 5389 5388 5165 4341
4351 4214 4514 5244 4232 5219 4451 4347 5218 5217 5216 4542
4231 4215 4343 4607 4340 5265 5148 4487 4217 4221 5146 5145
4452 5138 4353 5523 4483 4446 4574 5251 5252 5250 5247 5516
4786 4334 4333 4785 6464 6475 4705 5135 10005 4449 4947 6498
4925 4469 4413 4949 5997 4946 4944 4941 4939 4929 4930 4928
4194 4919 4918 4466 5144 32548 10534 23327 6476 4936 4934 4932
5478 4327 5143 4223 4390 4943 4923 4484 23328 4727 4572 5162
4150 5573 4942 4924 4922 4339 4886 4455 4453 5248 4323 6392
4512 4445 5595 4945 4938 4415 4352 4901 6093 23314 4427 33053
33203 4822 5553 6480 5481 5156 4395 10536 5537 4608 4447 10002
5524 5482 5783 5155 5139 4900 5246 4920 4716 5140 4857 4719
4948 4940 4921 5480 5137 4926 5142 6462 5463 5141 5249 4937
5163 5479 4718 5452 4220 4412 6043 4419 10004 4931 4486 4474
4530 4478 4454 4485 4565 4935 5173 4731 4717 5136 4933 4326
5154 5164 5888 4605 4927 5171 4737 6009 6451 5152 5477 4787
4784 4229 4450 4376 5476 5170 4410 6466 6218 4703 6463 5558
6235 5310 4704 4222 31946 23324 23318 23320 23319 23312 23311 23310
23309 9299 9268 9298 9300 7501 8401 9217 8606 8167 8162 8699
8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 8876 8878
8917 8958 9297 8970 8063 7480 8607 7795 8203 7565 7415 7447
7414 8208 7210 7312 8349 8842 8445 8159 7417 8841 8511 8867
8157 8959 8169 8928 8916 8843 6730 6710 6594 15926 9585 16953
707 30215 20641 32546 23330 23321 28643 33630 713 28688 16534 17866
17865 17714 17713 18410 18409 18408 18412 18411 18407 31190 17936 20198
30911 11023 12112 786 32134 22556 32131 23107 32321 32322 23109 32133
32137 32323 32136 32135 21746 21488 23326 16609 12192 708 33622 571
18866 26868 13216 28044 13170
586: 31243 31242 15303 23325 23313 9952 23316 13118 31135 21736 17283 27470
21777 26917 25140 21043 15800 23030 28223 667 20602 33625 712 30130
22230 31075 9864 32235 10424 20071 9988 33180 28669 28668 17680 29309
30013 30015 23751 15749 31624 33188 33187 31392 14334 14635 14451 33042
28788 14231 10603 11816 13265 13264 31578 33087 10561 10560 10559 14277
10539 31630 16625 9971 24627 19515 13000 13001 14795 1258 33052 28653
31735 10540 23323 477 23063 3132 9733 32132 23108 20083 18469 33178
14945 10076 13455 19522 31241 32011 32013 32012 16981 32491 31101 22002
29370 20325 23293 13487 23019 14473 19278 15423 18523 10558 10557 32814
20075 20076 33453 33451 33450 33452 33456 33454 33455 32395 23029 31809
23027 23028 19519 19520 32550 19516 32551 19518 32549 14435 19517 23322
23317 22969 33202 28787 23677 23329 23676 23315 22970 11111 31946 23324
23318 23320 23319 23312 23311 23310 23309 14565 10651 8659 8660 6863
8466 15926 9585 18463 32546 22115 23330 23321 33630 29794 17866 17865
17714 17713 18410 18409 18408 18412 18411 18407 17936 786 32134 22556
32131 23107 32321 32322 23109 32133 32137 32323 32136 32135 21516 14233
32515 23326 571 19463 15874
587: 22414 20378 13220 26615 26611 26616 13224 26612 26610 26613 26614 13222
13226 26618 13225 13223 13221 26617 26619 22822 20287 29127 20293 22059
21991 5586 27436 18862 26892 24604 26893 15392 31978 26890 15377 26894
26889 26891 28090 17888
588: 11116 30310 20087 23471 26729 25622 25574 25547 21178 21177 30728 9665
1829 1828 1336 1682 2739 2704 30750 14962 26905 33107 33106 11218
28674 32865 10969 26996
589: 18697 23456 23463 26812 26811 30158 13754 27158 31812 1262 1340 1496
936 1164 1689 1219 1394 29105 29121 5751 6604 29738 20675 20652
22946 31981 10223 11757 15860 17620 11740 32853 16621 19698 14777 19699
11749 33271 25309 19700 20117 18239 15728 28671 16307 17889 33144 24606
19157 15857 32503 33487 11741 24930 17613 33044 29327 22964 33077 32520
20118 15878 18238 33142 33147 31990 33486 33076 15734 9563 16962 11772
33080 33078 11782 11790 33473 33145 527 14264 17621 15856 17886 33143
590: 30755 13526 13567 12203 30236 30101 13097 31397 32909 33206 29386 29384
29377 29378 29380 29385 29382 29381 29379 29383 10918 15978 33549 10271
16120 27863 11196 29946 17290 12566 29426 25134 21057 13411 12678 12714
11266 18327 11703 18757 13844 20327 14209 18554 14375 15697 17745 33108
14611 14673 15181 15022 12939 32936 15123 15536 17560 22460 23758 15772
17784 16244 28093 21191 16718 26005 27535 16816 29857 29882 11885 19233
17176 17131 18095 28565 33109 18710 16323 24711 18879 19078 12805 12075
9898 33550 18112 14017 18488 19294 19817 19494 19656 19757 29922 25968
9432 25005 20571 25036 20774 32198 23217 12351 30330 22346 12272 33385
21637 22264 22884 21466 21373 21281 23127 24059 24064 23931 25732 24646
24673 27479 30091 28268 25122 26962 25702 27761 25448 10732 24972 25268
31061 26495 26233 27950 27286 11315 11383 9484 12032 11526 16466 16476
30616 13332 26097 26141 12514 10375 30511 16071 12619 12249 14916 19475
9839 30451 23037 32234 10425 28017 27374 18009 20070 28361 28125 32478
28465 24826 11732 32556 25027 18758 13697 27412 15933 16406 945 1247
1397 1559 20729 11564 24678 29092 20210 20207 20208 20209 17625 5909
4879 5549 31943 4655 31944 6443 6127 4814 5268 4962 5242 4880
5728 4650 4533 5197 5571 4349 5938 4430 4384 4368 5922 4968
4806 5529 9165 10044 9190 6867 7471 7479 6692 10042 7568 20145
7951 31517 8598 8597 8452 8051 7836 7215 7314 7182 10043 8749
7485 7670 8935 6771 31028 26885 24011 24013 24014 24012 24018 24015
24016 24017 30999 33315 33129 33127 33126 33130 33128 31000 14717 22602
21156 21153 21151 14721 21155 21152 21148 21147 22606 22603 22593 22592
22581 21157 21154 21150 21149 21146 14719 22588 22584 22589 22591 22583
22579 14720 22600 22590 22587 22597 22607 22605 22599 22596 22595 22585
22582 22577 22608 22578 22598 22586 21145 14718 22601 22604 22594 22580
21158 32555 18428 22920 22132 18662 14952 14693 14697 14694 14722 14695
14696 14709 14710 14708 14706 14704 14705 14707 14702 14698 14699 14700
14703 14701 14716 14715 14712 14714 14711 14713 28687 16508 16552 13234
9672 31183 27514 31298 30921 31593 31594 14887 14890 14888 14891 14889
32333 32332 12116 21764 16605 12185 19160 26837 20363 32263 33114 20724
31403 13173
591: 23597 24338 26806 26804 22756 1749 888 1534 1725 29144 29030 28951
5820 4754 4554 4288 5964 6349 6271 4544 6025 4206 4278 5371
6075 7953 8779 9289 9375 8014 6736 8719 8923 7248 7456 7590
8002 7612 7785 7657 7432 7531 7784 7790 6666 8751 8864 33004
10913 32115 33193 24637
592: 23483 23534 24445 26644 26722 26689 25531 25532 22645 18865 31977 29331
32854 32855 1038 1810 1042 1056 1357 1234 1389 948 1719 32398
32397 3860 3220 3687 3551 3604 31097 21987 4243 33054 12749 11172
18836 20644 19272 32881 24845 30839 8068
593: 23449 24469 26632 22657 9544 10871 1503 3716 5509 4426 8431 8430
7742 8432 8456 17264 14427 20630
594: 24610 15439 19155 29324 33477 9547 33479 9548 14834 33478 876 1723
935 3427 3731 3204 28947 22030 21964 29099 5716 4740 6085 7097
595: 23303 31546 896 1364 903 1310 24942 3883 22824 22825 22493 26906
10877 11275 15331 30992 22912 4103 5948 9213 8464 10481 18862 15943
596: 1239 1421 1931 29135 29258 29021 6402 4432 6225 9379 6696 6904
6871 7567 6837 32279
597: 17931 23278 12881 33025 27915 27550 29877 12012 16355 24702 19803 19788
19579 19573 21601 25728 11662 10783 26373 26382 27280 27305 26150 28402
1031 11575 4095 3426 3425 3548 4661 5925 6129 9120 8451 6841
8130 9119 8641 10178 16524 12120 28892
598: 13869 16693 18186 23598 24379 26754 25524 22725 25432 10679 26125 16197
26038 1385 11589 3477 2043 1976 29083 5591 6697 7030 9344 8462
7517 7031 20694
599: 33101 1601 3031 3597 3824 1926 3166 2111 5052 8148
600: 11554 11553 11555 31546 22414 14396 20378 14803 17597 1744 24942 3847
3848 28639 19429 21905 20580 20582 21904 20583 21906 20581 33244 33246
33243 33240 33242 33239 33241 13085 5568 4954 4506 32810 11551 11552
10480 10481 10482 10483 27167
601: 3404 2616 6255 4458 4535 5207 8557 8625 8168 8488 7495 8626
6640 32524
602: 31379 30665 13541 27847 22512 11203 16154 10905 25779 13397 12668 18340
33015 14222 14543 14380 14681 14489 32602 19015 14137 17548 15081 23814
15793 27913 28063 16237 18790 25996 16806 21886 17174 17452 18602 18846
18684 18731 16316 24698 12826 12071 9884 17970 19412 19799 22834 19491
19627 9704 20404 30123 12478 23212 30326 24299 21016 22869 24207 23964
25712 26049 26952 25220 27039 27251 11365 11486 11505 16460 12389 13319
30531 32036 11250 10846 28179 28478 16441 1695 1249 2513 2028 3012
2951 2287 4732 5754 4255 4256 6016 5379 4757 8695 7178 6624
7119 8278 8277 6896 7762 16505 31320 20883 11021 12098 31256 31411
603: 1738 29007 30751 6263 5313 4623 5533 8280 7910 8954 7538 7537
7486 18762
604: 13109 27877 33014 29279 14616 15173 27582 24694 18123 19796 23526 24383
24437 24441 24533 26678 26677 20788 21448 28254 27174 23947 30060 24265
27670 27810 10720 10669 30160 30163 30164 30167 30169 27252 27339 28405
13657 14763 22752 1021 11147 11406 11603 11606 14964 23016 3303 2054
3279 3858 3688 17715 15954 21937 32167 21931 9738 15481 15479 5720
6409 4158 6276 5213 4122 5201 4248 5200 4999 9064 8436 7096
6648 6839 7911 8352 9023 6731 6776 28232 23104 20676 18457 22952
10336 28737 22894 30919 22891 20125
605: 9802 33199 15213 9745 13538 13530 27608 27647 30244 30099 11418 31676
21585 10272 10249 11191 11179 24080 24076 17310 17362 17346 17319 17348
17325 17358 12573 25161 13432 18303 18316 12861 13888 23662 20384 20382
14534 15696 14585 14592 15158 15156 15137 15002 14978 12904 12896 12926
32305 15120 20074 19028 17520 17561 22442 23759 15291 14464 15292 23760
17002 17791 17765 16277 29826 29819 28101 26003 32371 32365 27549 27589
16831 15905 29895 20488 25984 25975 11920 11956 11988 11911 11975 11954
19228 17467 17147 17125 17136 17124 17243 18034 28552 28582 28576 28574
28563 18674 18704 16309 16395 16338 24754 24770 24723 18913 18961 18943
18964 18897 18944 18926 18903 19125 19118 19122 12811 12807 9877 18139
18228 18212 18101 14030 14055 19317 19860 19831 23498 23521 23625 23538
23592 23611 23436 23423 19610 19607 19558 29927 20022 25006 24999 24455
24555 24416 24351 24549 24417 24352 24552 24419 24559 24468 22974 26744
26709 26735 26736 26731 26684 26675 26734 26755 26711 26668 25537 25877
25884 30135 22655 22799 22698 22683 20809 20769 32225 32228 23200 23232
30367 22334 22329 24302 33433 33434 21648 21632 22569 22288 22874 21449
21426 21427 21402 21340 21339 21297 21306 21256 21255 24200 23248 23249
23139 25913 29488 29491 29968 27195 27184 23985 25179 30186 26089 25817
25825 30056 24278 30041 25129 30035 26982 25259 27684 27678 27704 27662
25455 25470 13035 13045 10695 10740 10660 27087 13749 13739 27151 31057
26465 26487 26422 26444 26398 26289 26244 26245 26392 26335 26235 26302
26320 26316 26371 27927 27263 27245 9483 11530 11465 11517 29759 29791
29751 29771 30650 30600 12402 13352 26127 16178 16214 26152 24583 17924
23681 16078 14892 28024 27990 27989 27336 28424 28418 28454 28433 28306
28297 31797 14755 15591 20836 31796 13679 13664 24806 19288 23403 31236
31237 24835 24834 16412 29732 16425 29689 16430 17750 988 1538 1315
1313 1307 1169 1170 1581 1181 923 1110 11590 3834 3975 2709
3046 3524 3527 3628 3331 2412 3047 23401 31915 23400 24913 23399
23402 24914 33007 32088 29196 29197 29199 19940 29195 29315 29198 28955
15855 28228 11221 4564 6254 5928 6068 5937 5653 4563 5320 5060
5311 6000 6111 4276 5059 4597 6313 4257 5312 5302 5613 4262
5521 4670 6524 6133 5782 5815 8353 9195 7841 6912 7918 7687
6861 6834 8908 6581 6950 9100 9099 9194 33236 33237 23745 16032
16033 16031 28342 29177 12748 32525 24951 32526 16636 25021 24023 27014
23666 19154 20672 22980 13237 28870 28328 28327 28758 28772 28736 28690
28703 16533 16564 16538 12440 31217 31194 30906 31021 12150 12964 31432
31450 13166 15448 18438 24628 11770 17880
606: 26607 1758 1125 1630 1013 3799 2945 2454 2670 3798 2350 2149
3927 2573 22008 29031 23888 31952 4854 4855 6493 10958 7774 8720
6667
607: 1332 869 1080 28884 21557 28982 29247 30204 20188 20187 15572 3592
2306 2995 3342 3659 2814
608: 31985 1011 18537 21118 6080 6119 8019 8174 7224 8814 3254 3955
2687 2714
609: 13534 30276 30100 17223 11604 13141 13099 30669 32310 31903 25777 25778
21047 20380 14624 14668 15104 15540 22480 22469 15806 15766 28099 28214
16881 15912 25978 17476 17091 18078 18090 16345 19092 19069 9901 19375
19295 9680 30591 25892 20601 12354 12334 30349 12284 25921 25926 25185
27509 25816 25784 25674 27813 24957 24958 31060 27260 9534 30628 26117
12623 12844 10841 27319 30769 30768 10006 24932 24918 24917 26551 24919
14389 32416 28625 9676 9771 28622 28623 994 1250 1666 1556 28624
28626 27465 3896 1847 3875 3233 2605 2604 2392 2892 1997 2391
16030 29041 28960 28959 15645 33600 24802 29224 12425 12424 5237 22987
24321 5676 5695 5601 24322 4803 5787 9010 6907 8471 7352 6823
6926 31346 28627 28628 16548 31226 14430 19722 28629 20590 21484 33629
10047
610: 1409 1650 1992 3114 3695 2846 3492 29039 5079 5634 7856 8855
8533 9323 6762 8989 18449 31784 32236
611: 23485 24384 26628 25582 1222 1116 31814 3571 3095 2405 2804 2086
29076 22038 31103 4543 6363 4228 5373 5378 4736 4906 8254 7756
7958 7327 8995 8978 21685 9437 23673 20722
612: 11554 11553 11555 11558 11559 31546 14396 20378 31617 3622 3522 2662
28639 22493 28928 29006 21904 21905 20580 20582 20583 20581 21906 33240
33242 33241 33239 13085 6388 5084 6008 4624 5772 8925 6782 7977
9399 11552 10480 10483 10482 10481 17617
613: 13568 23325 23313 9952 23316 13105 815 759 31135 17283 22061 26917
25133 21071 15785 23030 33625 33618 465 25039 22284 31073 33180 28669
28668 17680 29309 30013 30015 31392 14451 11816 13265 13264 31578 17603
10474 10561 10560 10559 14277 10539 24627 16625 9971 19515 13000 13001
14795 871 907 906 905 1403 908 872 1825 1398 1402 1126
1545 1401 1153 1456 1251 1544 1376 1414 1527 1326 1382 1354
1494 1399 1370 1361 1341 1476 1587 1504 1817 1623 1264 1639
1417 1653 1543 1651 1590 862 1233 1377 1715 1400 33052 10540
23323 477 23063 2641 3109 3386 3273 2964 9770 2014 2319 2635
3299 2144 2961 2963 2013 2015 2898 10538 2993 1853 2061 3272
2105 9769 2485 2214 2718 2135 2962 2142 2617 2139 2676 2490
2257 2965 2543 2966 2289 2277 3328 2134 2143 1928 2724 1939
2130 2491 2967 1965 2140 2308 2128 2313 2310 2193 1998 1850
2480 2131 1912 2192 1851 2592 2241 2212 4052 1849 2316 2245
1996 4048 2997 2968 2118 3086 2133 1839 2129 2297 2486 2176
4047 1894 2479 2172 2076 2727 1947 4046 2208 4050 3066 2141
1892 1843 1913 2242 2179 1937 2655 2251 2290 2749 2132 3023
4032 2373 4069 1896 2492 2075 2279 2299 2171 2577 2320 2200
2155 2258 2180 4056 2174 1891 4057 1966 4071 1865 4037 4033
1842 2000 3073 1863 4049 2191 1899 2779 1943 2712 2234 2383
9733 32132 23108 20083 18469 14945 10076 13455 19522 32011 32013 32012
16981 20107 20325 23293 13487 23019 14473 19901 13486 15423 18523 10558
20075 20076 33453 33451 33450 33452 33456 33454 33455 32395 23029 31809
23027 23028 19519 19520 32550 19516 32551 19518 32549 14435 19517 23322
23317 10537 21855 10535 23677 23329 23676 23315 6270 6332 6418 4324
4328 4354 5153 6314 6490 4301 4783 6465 4472 4342 4859 10003
4218 4726 4863 4216 4230 4448 4741 4325 4337 4219 4573 4329
4411 4473 4330 4338 4213 4720 4513 4511 5389 5388 5165 4341
4351 4214 4514 5244 4232 5219 4451 4347 5218 5217 5216 4542
4231 4215 4343 4607 4340 5265 5148 4487 4217 4221 5146 5145
4452 5138 4353 5523 4483 4446 4574 5251 5252 5250 5247 5516
4786 4334 4333 4785 6464 6475 4705 5135 10005 4449 4947 6498
4925 4469 4413 4949 5997 4946 4944 4941 4939 4929 4930 4928
4194 4919 4918 4466 5144 32548 10534 23327 6476 4936 4934 4932
5478 4327 5143 4223 4943 4923 4455 4484 23328 4727 4572 4150
5573 4942 4922 4339 4886 4453 5248 4323 6392 4512 4445 5595
4945 4938 4415 4924 4352 4901 6093 23314 33053 33203 4822 5553
6480 5481 4395 10536 4427 5537 4608 4447 10002 5524 5482 5783
5156 5162 5155 5139 4900 5246 4920 4716 5140 4857 4719 4948
4940 4921 5480 5137 4926 5142 6462 5463 5141 5249 4937 5163
5479 4718 5452 4220 4412 6043 4419 10004 4931 4486 4474 4530
4478 4454 4485 4565 4935 5173 4731 4717 5136 4933 4326 5154
5164 5888 4605 4927 4737 5171 6009 6451 5152 5477 4787 4784
4229 4450 4376 5476 5170 6466 6218 4703 6463 5558 6235 5310
4704 4222 31946 23324 23318 23320 23319 23312 23311 23310 23309 8162
8699 8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 8878
8876 8917 8958 8970 7480 7795 7447 8208 7210 8349 8842 7415
8445 7414 8159 7417 8841 8511 8867 8157 8928 8916 8843 6730
6710 6594 15926 9585 707 585 32546 23330 23321 16503 17866 17865
17714 17713 18410 18409 18408 18412 18411 18407 17936 32134 22556 32131
23107 32321 32322 23109 32133 32137 32323 32136 32135 23326
614: 14723 13123 32406 22084 11281 11280 11279 14753 17637 15590 27134 32532
17429 22570 17430 15629 14761 14760 23014 13965 32083 32530 17334 25769
21033 12925 24740 12815 12780 18150 19852 23565 23430 19578 19536 19748
24342 24409 24541 26679 25609 30132 22742 22637 22747 22677 21459 21327
21243 27191 23959 25840 26585 30033 27754 31046 26453 26309 27208 12041
16169 27337 28395 10977 33534 23175 10334 13263 1518 1672 3525 2675
3992 3714 3982 2331 3276 2376 3981 2985 3378 32973 29312 28931
32573 27136 15756 15351 10866 10974 4652 5673 6116 6121 6115 5386
4653 4184 9339 8480 7611 8955 8639 9746 9747 24854 31592 11239
18472 23669 20628 15938 9915 16938 29805
615: 33195 17944 33588 18526 32474 13576 13555 13569 13236 27606 30103 13100
13093 13144 13119 32904 32910 32902 32916 24115 17564 27833 17563 33506
9772 17262 10254 10260 10293 10289 10280 21683 16110 30301 30290 29575
29576 11211 11183 29940 24090 24088 25896 16130 29592 16152 16149 17312
17291 17419 17299 17296 17322 17404 17295 17369 10901 10887 10893 12565
12554 12564 24236 25152 25137 21048 13426 13389 13367 13387 13428 13424
13415 13433 12656 12669 12658 12665 12655 12695 12697 12706 18350 18338
18367 11730 13925 11722 13933 13903 13919 13927 13857 21682 13909 13938
11714 13875 13899 13870 13891 10925 31509 14198 14190 14203 18553 18571
18552 14539 14530 14524 14544 14531 14371 14377 15687 15692 15688 15683
15685 14605 14589 14597 14686 14499 14513 15147 15210 15153 15038 15051
14997 12945 12912 12890 12887 32294 32630 32616 32609 32614 15115 15111
15103 15121 19019 19023 19006 19024 19013 19025 15530 15537 14150 14129
14139 14141 19166 17534 17527 22444 22420 22437 22440 14461 23802 23795
23780 15281 20226 15769 15798 15804 17024 17012 16996 16997 17006 17026
19933 15614 15617 17831 17780 17778 17813 17799 17834 28060 16248 16264
16252 16281 16262 16287 16275 16273 21196 16788 16771 16689 16682 16785
16712 16769 25991 16663 16669 16869 32350 32369 27599 27537 27600 27591
16801 16827 16803 16874 16896 16889 29836 29847 29839 29835 16877 29854
16875 15899 15902 20489 11989 11961 11905 12006 11893 11921 11971 19232
19207 19212 21872 21867 17177 17187 17173 17163 17190 17192 17478 17457
17460 17448 17470 17451 17146 17152 17098 17141 17090 18589 18605 18607
18597 18606 12735 12733 18061 18092 18064 18039 18028 18046 18031 18033
28516 28571 28545 28604 28583 28608 28561 28575 28588 28538 28539 28514
18692 18695 18721 18720 16325 16364 16396 16341 16352 24767 27411 24751
24699 24718 24697 24743 24689 24693 24708 24832 18965 18885 18889 18931
18983 18891 18985 18899 18941 18989 19101 19117 19070 19126 19067 19039
19060 19116 19061 19123 19080 12818 12809 12817 12074 12084 9883 9897
9891 9874 9899 9890 18204 18207 18136 18173 18226 18222 18127 18159
18107 18220 13997 14028 14010 14054 14015 13982 14058 14039 14034 14024
19379 19413 19384 19377 19367 19386 19389 19376 19340 19311 19339 19306
10075 19873 19813 19800 19826 19795 19791 19810 19854 19484 19504 19650
19666 19668 19581 19692 19569 19590 19637 19631 19759 20482 29923 20481
25939 20459 25956 20012 20015 22571 24035 25008 25009 20972 20564 20548
25057 30107 25048 30114 21716 21724 20786 32214 32183 23186 23194 23189
23198 23236 23218 23185 12352 30345 30361 30317 30366 22369 22312 22324
22325 12288 12307 12285 33407 33386 33370 33435 33393 33360 33425 33428
21690 21621 21619 21622 22294 22254 22843 21478 21414 21450 21463 21410
21437 21447 21443 21394 21305 21388 21289 21380 21320 21323 21334 21354
21363 21385 21338 21318 21288 21335 21294 21348 21284 21214 21237 21269
21262 21231 24193 24210 24201 24192 24183 24202 24173 23264 23161 23163
23129 25912 29961 29962 27198 27179 24004 23961 30173 25727 30182 26067
26068 26076 26061 24652 29992 25869 24655 25868 23173 30052 30050 30077
26602 26597 24261 24295 24289 24296 24286 24258 24264 28292 25781 25786
25782 28843 26977 26927 26943 26975 26978 26983 26966 25231 25216 25677
25212 27692 27667 27729 27776 27721 27777 27791 27717 25328 25472 25314
25456 25312 25459 25489 25442 25440 10754 10674 10782 10689 10780 10710
10792 10706 24961 27070 27085 27068 27031 27057 13750 25282 25266 25294
31066 31090 26458 26442 26471 26520 26430 26209 26327 26195 27955 27958
27300 27265 27295 27223 27293 27219 27210 9452 9516 9482 11483 11464
11531 11519 11469 11541 11506 11482 11539 29778 29770 29769 29763 16468
29789 30614 30608 30679 30601 30594 12405 12406 12388 13311 13345 26126
16229 16227 26111 26112 12525 12509 24574 10366 10385 30516 16041 16077
26044 26035 26025 16060 26012 16056 16064 12599 12611 12021 18633 18635
12618 12624 12256 14899 28191 28008 27318 27351 27355 27359 28409 28381
28410 28399 28131 28438 28491 28476 28458 28300 28322 24812 13715 13724
13655 13723 13713 13700 13682 15751 31626 15742 24024 16408 16403 16400
16422 29693 32268 33458 9668 21684 17583 1093 11599 11635 4054 4053
22141 22151 20206 28923 32816 5192 5929 9008 8236 8682 7807 7347
8866 30827 31029 17567 28234 31508 32324 28765 28711 28755 28771 28777
16563 16576 16541 16549 16535 16511 12439 32492 31169 31161 31139 31157
31210 11645 31292 31305 31302 19684 20876 20199 20004 31778 21506 23105
30893 30951 30944 31604 31024 24145 11016 11033 12099 31269 12145 12950
12959 12974 12978 12969 12986 10606 31777 21743 21769 21748 21483 21485
16611 16601 12190 12182 26831 26849 26840 26848 26862 26844 26830 21681
31425 31419 12754 13172 13175 13183 13181 21139 14802
616: 33046 22935 10215 31712 23612 24473 26623 25556 22679 31786 11130 9443
10427 22821 27528 27529 10945 14287 33470 28338 28339 32385 26045 1078
20058 20057 24935 32139 28868 22034 18517 33088 32469 12998 32991 10531
29231 29232 6145 4185 5749 6090 6147 8397 8073 8509 8998 9095
9180 9094 8915 8997 6735 7732 17435 29230 16644 17076 10552 15295
9926 9925 13231 9924 22954 27461 29154 29156 20611
617: 31945 873 870 1595 959 3705 2701 3291 2482 2933 1927 2627
3292 1949 2060 22017 30657 6399 5863 4463 6204 4489 5603 6327
5359 9183 9182 8007 8111 8581 8150 6720 8936 6632 8582 6598
6680
618: 14764 11554 11553 11555 31546 29412 1045 979 24942 2606 3028 3750
3749 2607 3493 3006 28901 30193 28968 21904 20580 21906 20583 20581
21905 20582 33240 33242 33239 33241 30394 20078 5105 5625 8883 7933
7932 7810 7982 32810 11551 27445 27436 24600 20116 15888
619: 30231 29276 14351 17731 21890 21895 20962 12828 13777 19333 9689 15748
12358 12293 27472 27496 25705 27738 16490 29752 30613 26147 30527 10835
28145 28118 3666 4051 4030 2272 2271 2063 33189 5910 4332 4387
6141 6387 6320 6029 6264 5385 4367 6329 18813 9732 32113 33177
18830 18829 32885 31537 13049 31230 31323 20864 20589 13162
620: 23532 24522 25619 1683 4020 3183 2207 2650 3040 2850 2851 3053
3036 2569 3572 2281 1988 1970 3070 2246 2094 2720 1885 4841
5861 9086 8017 6977 7775 6794 7436 9429 7228 7233 8481 6976
15476
621: 22815 10867 20536 24336 25621 9576 9577 19998 21692 10007 20157 28784
33482 1678 1092 1428 10511 3758 2782 29311 29102 33513 32982 29234
5033 33089 16944 14868 11792 10486 17263 33329 9407
622: 32587 31497 9914 27135 21110 33569 31548 10256 25904 17383 13422 13361
15653 15140 14982 20142 23768 14455 16993 17845 17773 27890 28064 29879
12015 19256 17092 28550 24741 9900 18179 18102 18178 23610 23442 23550
23623 19532 31127 24448 24421 24413 20391 23296 25663 25647 25890 21807
22633 22721 22699 20781 32211 22341 33420 21689 21412 21403 21473 21299
21386 21219 21216 23937 23926 23975 23991 23962 23936 23949 25187 27506
30059 30075 25115 25852 25844 25826 30040 26973 26921 25258 27715 27705
27756 27812 27782 10770 10718 10730 27077 27038 27046 13742 13761 26505
26490 26500 26329 26305 26315 26276 26395 26385 26275 26306 26390 26258
9497 13342 16200 12222 11247 10833 27311 27389 27325 27331 27420 14968
14301 18376 31773 14061 17712 30016 15297 13695 13722 30551 1816 11611
27530 3211 1832 31852 21956 21958 21957 21955 33251 24106 32954 24849
16014 26541 6372 6333 7332 9255 6793 31015 32330 26545 26544 26537
25019 17701 16036 20102 13285 28804 28717 28774 31188 18802 30882 25345
25343 25344 25341 25340 25342 24566 11039 11022 11011 12110 12141 12111
12089 12956 30558 13200
623: 27439 32868 10208 22613 18256 17559 18123 19796 19671 24998 24997 24383
24533 20430 25558 22693 22727 20805 20788 23947 27252 28370 11129 15307
27011 10530 14164 15757 14763 22752 1071 11406 33563 11606 11602 10639
33000 2721 3908 13012 17715 33258 14099 17943 17890 16039 15954 30466
30465 30464 15953 27527 20320 32579 32578 32167 32545 15072 11288 33173
12487 21931 11139 12596 15481 15479 9738 5908 4178 4706 5299 5298
4767 23336 10336 32665 28748 11044 31276 17682 33190
624: 1123 1105 1480 2760 2640 6512
625: 22614 28807 1768 28953 6192 8912 8567 4021 2902 2385 3501 3309
2903 3051 3067
626: 17218 32247 33070 13551 32396 27648 28241 30260 30239 11687 11686 18406
32926 32903 25339 10497 14414 33571 18655 23099 32103 32102 11294 10297
10266 11215 11174 29957 12594 17293 17392 10899 31632 13392 12661 12701
31288 18344 18354 24857 13928 12850 13943 13905 13937 11721 11729 13908
14211 31882 24148 20214 10936 15957 14538 15667 15707 17733 25308 14680
12930 32610 15100 29221 14157 17508 17495 22418 22467 23765 23782 23783
23819 15287 17005 19927 15613 17816 17785 27900 27907 27883 28079 16794
16776 16784 16846 32366 27546 15903 20517 11891 19244 17183 17464 17106
17244 18037 28558 28515 28513 15238 16385 16367 16321 16335 24691 24724
18883 18881 18933 18932 19075 19100 12773 12054 18113 13989 14048 14025
14040 19368 19793 23713 23596 23492 23572 23469 19687 19991 25941 25966
24527 24471 24397 24466 24370 9694 26650 26782 25646 25645 30113 12467
23192 12357 30354 22320 33390 21664 22289 22851 21428 21438 21471 21341
21314 21293 21287 21383 21257 21230 21282 23253 23256 23268 23146 23128
23144 29480 27189 30188 25189 26063 27477 25101 25846 30086 25843 25823
25080 26947 25668 25213 25219 27672 27683 27774 27816 25453 25461 25429
25471 25457 25475 13030 13040 11679 11671 13025 11666 13023 10682 10764
10747 10767 10745 24980 27073 27043 13775 13740 13741 26488 26428 26425
26468 26467 26519 26393 26274 26377 26314 27975 27297 26396 9475 9522
11542 29783 30619 13314 16212 16216 26107 10401 26013 16066 12603 10973
12220 10840 27018 17860 27988 28030 27321 27357 17927 28364 28368 28357
16037 30723 28482 28316 31502 10929 13830 20088 17591 15367 18372 26887
20043 13648 13687 14450 33357 20935 23721 22202 29186 29724 16442 13611
30557 23665 27457 14884 14878 11563 33614 2894 3801 1910 14452 22154
31949 11639 11436 33317 4503 5230 5232 5231 9178 8669 9179 6668
10856 21539 21540 32476 31867 24828 32006 25380 20647 25024 31393 31394
33304 31866 14066 19280 9596 28753 28768 16553 12441 12462 24921 31174
31151 21144 30902 30936 30916 29212 24146 22203 11037 12090 12138 12104
32972 31439 32037 28047 31553 13185
627: 10188 26608 32646 32645 17282 15190 12922 28209 23489 23626 24410 24440
26799 22798 22739 22630 23132 23934 16182 26990 27353 22561 28341 32953
31338 990 924 1175 961 1573 1194 1405 10190 1856 32642 21204
21205 21999 21998 31478 26916 6426 5054 8684 6957 6615 8722 7820
7046 7818 7819 7779 6638 31647 24110 23398 11223 26546 25020 20625
9588 20712 15925 15581 23024 23023 28501 17938 19465 19466 19464 17933
19726 32562 17937 22820 18763 11028 12119 9553 17883 10216 18776 18774
18775 18772 18773 18771
628: 13554 23325 23313 9952 23316 13089 759 31135 17283 22061 26917 25150
21077 15790 23030 478 667 33625 33623 33615 25047 31062 33180 28669
28668 17680 29309 30013 30015 31392 14451 11816 13265 13264 31578 17603
10474 33620 14277 10539 16625 9971 24627 19515 13000 13001 14795 871
907 906 905 1403 908 872 1825 1398 1402 1126 1545 1401
1153 1456 1251 1544 1376 1414 1527 1326 1382 1354 1494 1399
1370 1361 1341 1476 1587 1504 1817 1623 1264 1639 1417 1653
1543 1651 1590 862 1233 1377 1715 1400 33052 10540 23323 23063
2641 3109 3386 3273 2964 9770 2014 2319 2635 3299 2144 2961
2963 2013 2015 2898 10538 2993 1853 2061 2105 9769 3272 2485
2214 2718 2135 2962 2142 2617 2139 2676 2490 2257 2965 2543
2966 2289 2277 3328 2134 2143 1928 2724 1939 2130 2491 2967
1965 2140 2308 2313 2310 2128 2193 1998 1850 2480 2131 1912
4052 2192 1851 2592 2241 2212 1849 2316 2245 1996 4048 2997
2968 2118 3086 2133 1839 2129 2297 4046 2486 2176 4047 1894
2479 2172 2076 2727 1947 2208 4050 3066 2141 1892 1843 1913
2242 2179 1937 2655 2251 2290 2749 2132 3023 4032 2373 4069
1896 2492 2075 2279 2299 2171 2577 2320 2200 2155 2258 2180
4056 2174 1891 1966 4071 4057 1865 4037 4033 1842 2000 3073
1863 4049 2191 1899 2779 1943 2712 9733 32132 23108 20083 18469
14945 10076 13455 19522 32011 32013 32012 16981 20107 20325 23293 13487
23019 14473 19901 13486 15423 18523 10558 20075 20076 33453 33451 33450
33452 33456 33454 33455 32395 23029 31809 23027 23028 19519 19520 32550
19516 32551 19518 32549 14435 19517 23322 23317 10537 21855 10535 23677
23329 23676 23315 6270 6332 6418 4324 4328 4354 5153 6314 6490
4301 4783 6465 4472 4342 4859 10003 4218 4726 4863 4216 4230
4448 4741 4325 4337 4219 4573 4329 4411 4473 4330 4338 4213
4720 4513 4511 5389 5388 5165 4341 4351 4214 4514 5244 4232
5219 4451 4347 5218 5217 5216 4542 4231 4215 4343 4607 4340
5265 5148 4487 4217 4221 5146 5145 4452 5138 4353 5523 4483
4446 4574 5251 5252 5250 5247 5516 4786 4334 4333 4785 6464
6475 4705 5135 10005 4449 4947 6498 4925 4469 4413 4949 5997
4946 4944 4941 4939 4929 4930 4928 4194 4919 4918 4466 5144
32548 10534 23327 6476 4936 4934 4932 5478 4327 5143 4223 4943
4923 4484 23328 4727 4572 4150 5573 4942 4922 4339 4886 4455
4453 5248 4323 6392 4512 4445 5595 4945 4938 4415 4924 4352
4901 6093 23314 33053 33203 4822 5553 6480 5481 4395 10536 4427
5537 4608 4447 10002 5524 5482 5783 5156 5162 5155 5139 4900
5246 4920 4716 5140 4857 4719 4948 4940 4921 5480 5137 4926
5142 6462 5463 5141 5249 4937 5163 5479 4718 5452 4220 4412
6043 4419 10004 4931 4486 4474 4530 4478 4454 4485 4565 4935
5173 5171 4731 4717 5136 4933 4326 5154 6009 5164 5888 4605
4927 4737 6451 5152 5477 4787 4784 4229 4450 4376 5476 5170
6466 6218 4703 31946 23324 23318 23320 23319 23312 23311 23310 23309
8162 8699 8482 8879 8875 8872 8877 8873 8971 8874 8161 8605
8876 8878 8917 8958 8970 7480 7795 7447 8208 7210 8349 8842
7415 8445 7414 8159 7417 8841 8511 8867 8157 8928 8916 8843
6730 6710 6594 15926 9585 707 585 32546 23330 23321 33630 713
16507 17866 17865 17714 17713 18410 18409 18408 18412 18411 18407 17936
786 32134 22556 32131 23107 32321 32322 23109 32133 32137 32323 32136
32135 23326
629: 33105 30663 9779 33286 13542 13563 13560 13604 13536 15662 12195 30271
30257 25067 31544 30104 13107 31955 13088 32748 9799 32319 27831 27825
27826 12427 9813 9777 18462 15569 30307 30306 32574 10292 30294 16107
30297 29572 16113 29565 12722 29950 24095 25897 17397 17331 17316 10903
12560 12555 12544 32126 25754 25141 21042 21050 13363 12683 12720 20823
20822 18364 18368 13849 13863 32649 14197 20385 18575 18570 18542 15231
14541 14379 15690 15689 15720 15680 14608 17055 14509 14506 15191 15144
15143 15030 15041 15042 12943 12886 33526 32289 32617 19017 15538 15559
14140 14121 20938 19462 17549 17526 22456 22459 14462 23815 15286 23807
12429 15774 15815 17013 30973 19932 15506 17811 17812 28066 28068 16299
28089 21195 16698 16665 16847 32359 32378 27568 27601 27595 16830 29851
20520 20502 11953 11914 11899 11942 11915 19208 19240 21874 21879 17175
13013 17453 17130 17140 18604 12743 18049 18096 28527 28526 28606 28532
18677 33532 16311 16354 16343 24720 24769 18957 18907 18928 18880 19057
12761 12819 12816 12076 18185 13979 13996 19396 19372 19328 19312 19783
19828 19876 23454 19509 19598 19629 19689 19600 19640 25945 20455 24547
9691 20984 20398 20561 20541 26649 26705 25568 25590 33331 25043 25876
25050 30128 25028 22783 20784 20598 20800 32194 23182 23230 30363 30338
22327 22326 22306 22376 12291 33381 33369 21650 21641 21646 21651 22232
22231 22870 21367 21235 23262 23147 24056 25935 23960 23923 25746 25713
26082 25797 24666 27474 27493 25108 30093 24267 24294 24269 28290 28282
30043 26923 25203 25686 25682 25669 27694 10464 27773 25451 10721 10723
10719 10665 10793 10791 13746 13747 25287 25280 31091 31068 26477 26482
26361 26334 27966 27303 27221 27225 27240 9450 9520 11507 11484 29753
29750 29758 30597 30611 12376 12383 13290 16225 12510 12532 10406 30509
26028 16058 12615 12253 14940 14911 10829 21002 28001 28033 27360 27326
28363 28120 28441 28437 28426 28299 13670 21166 30734 10653 31662 11136
29728 29694 10346 27164 27163 19283 15889 1157 1604 1762 1736 1160
1081 32087 14552 14879 11570 11594 20229 3665 2861 3503 2697 3660
3655 22146 9734 31707 31708 32937 16723 20267 12489 13621 24020 21704
10150 14060 11311 9916 33134 28728 28763 16583 16522 16565 16502 12448
12436 31144 31201 31186 31810 31322 32403 21507 21520 30918 30886 21587
30687 23640 23641 20587 11050 31268 31255 12108 31253 12155 12977 21761
15587 16587 12164 26841 31424 31447 33521 13159 10218 20197 15445 11789
15583 15405
630: 17706 32387 1298 1217 1591 1776 2869 3909 18539 30588 29282 28949
30835 31467 21003 5594 4667 5652 5651 5650 5004 4110 6262 6167
5003 5005 5214 4109 6069 7678 8658 7213 9350 8143 7879 7878
6761 8769 7677 8325 6740 6849 8156 8155 9093 6955 7340 9076
8944 8154 8142 9177 7877 8419 8721 7709 7320 7405 9417 8993
8248 8727 8499 8172 32504 11784
631: 12215 32925 10294 27856 25901 17309 20381 15684 22451 17014 15525 16849
32356 27559 11977 17121 18053 28581 18901 19051 20476 25007 24346 20986
25642 30318 22359 33379 21603 21333 21254 23266 24060 29988 29995 25856
25128 26459 26225 16195 12601 28133 28873 22817 13672 16432 20084 1537
2064 2085 2500 4801 5871 4800 7212 9377 8975 31880 10547 32078
20670 10153 16676 28716 30949 20963 29974 13184 33484
632: 19324 1300 1633 3591 2770 3959 2334 24571 33339 4360 6456 5622
4865 4728 6532 4320 6158 6107 4729 6534 5369 6533 6294 5860
5367 5368 7497 8329 8330 7201 8583 8104 7274
633: 30490 9787 13546 32703 31872 32702 32704 31904 27618 32743 32693 32741
32738 32689 32695 32745 32747 32697 14342 11055 14340 30991 29474 29473
29471 29469 29464 29461 29468 29467 29472 29463 29462 29465 29470 29466
21561 21565 21566 21564 21562 21563 21567 17222 15761 555 31493 16973
15947 19951 11292 10253 33349 17330 31287 13877 13929 22533 23036 20213
19778 19777 13005 19775 15706 15198 15201 14990 14987 12919 12924 12914
14798 17503 22453 23776 17770 19950 19943 19941 19945 19942 19949 19948
19946 19944 19947 27918 16777 33613 25983 11972 17159 31491 31492 17155
17119 19973 19967 19966 19971 19969 19962 19970 19964 19968 19965 19974
19961 19972 19963 19956 19955 19953 19952 19954 12726 18051 28569 18737
16384 18906 19111 18213 18231 14013 14047 23714 23502 19779 19670 19604
19681 19649 29928 20011 20009 24565 22976 17869 17868 26770 25660 25049
22729 22373 21598 22296 21392 23160 29477 23917 23987 23992 23955 24642
29994 25815 25109 25839 24259 30155 27697 27767 25473 11663 13042 10675
13738 31059 26427 26263 9493 12523 16075 12259 28032 15366 15364 32729
22522 22516 22515 22517 32736 32734 31683 17946 32877 33498 28331 33502
14271 32827 14071 33530 32828 13661 32393 26162 10161 10162 10155 10156
10157 10160 10159 10158 10163 10154 31751 31750 32876 20185 28330 17954
17950 17951 17956 17953 17949 20158 13015 31753 31752 31748 31749 31755
31754 31756 31766 31765 31763 31764 31757 31758 31762 31761 31760 31759
29268 31494 32513 31490 16427 21001 17234 17239 15949 17948 17955 22090
11832 11831 10477 32956 32957 32875 17952 32874 17947 33503 33504 33500
33499 22098 23913 23911 23907 23905 23906 23904 23902 23901 23903 11835
11834 11839 11842 11841 11833 11840 11838 11849 11836 11846 11845 11848
11843 11851 11847 11844 11837 11850 17945 32878 33501 20159 33497 16931
21576 21575 21574 9853 21211 11449 17659 32460 18651 14445 14443 15451
14444 10440 10441 14270 15434 14805 15406 24595 13736 24636 9441 28860
15731 15474 14281 15730 29333 32844 32502 1787 1767 874 32438 23074
14292 33281 11584 9797 11630 32871 3988 3280 3989 2729 2918 2716
3284 1938 2324 1905 15927 3643 3885 23334 4075 2239 3111 3821
24853 3502 3670 2237 2222 2295 3447 2623 3625 2189 2044 3884
3446 2540 3950 2426 3344 2571 2939 3504 3990 2071 2590 2432
4026 2019 3194 3805 2980 3680 2188 17237 17240 17238 31489 20222
15630 31888 31889 22143 13282 14769 28995 28963 22004 21968 28964 20282
32680 21969 17649 13793 21902 32682 32728 17629 32701 31871 16965 15945
16966 11070 11080 16964 16968 16967 15950 17609 11082 11081 11073 11071
11072 11084 11083 11085 11086 11088 11087 16974 15948 33115 9730 33116
31877 27442 19468 19467 9731 9840 9591 31885 21573 21571 21570 21572
21569 21568 14824 14339 23073 21900 21899 21898 21901 32726 32681 32635
13479 21903 32737 32686 32744 32694 32735 22526 22520 32685 22525 22519
32732 22521 32683 32727 32730 10979 10978 29628 29632 29630 29629 29633
29631 32244 21680 6067 5819 6431 5729 5918 5921 4797 4798 30397
5203 5555 5421 4799 5920 5422 5424 5919 4796 5917 6288 4244
6175 4795 4208 5058 5554 5904 5906 5971 5647 5849 6176 5423
5556 5443 5053 5818 5014 6177 5206 6194 33348 10194 8574 9266
9267 6719 7909 6707 8942 7708 7704 9265 6639 9331 6721 13446
7875 7153 23005 9330 9332 30192 8526 9333 9133 7812 32533 9011
6626 7604 7216 30662 30661 8217 8604 8216 7754 8941 7373 7834
7603 7602 9232 8724 7606 7601 7600 8129 7179 7607 7067 8527
8218 7887 7817 7259 7605 8324 7099 8985 11658 11115 10195 10198
10197 10196 17241 31014 13008 13009 19362 13014 20171 31387 33496 32580
13017 20165 20166 20164 20160 20161 20162 13016 20163 20169 20170 20168
16654 20172 20173 20175 20174 20176 20180 20177 20178 20179 20181 20184
20182 20183 13778 13779 21537 22749 30727 13733 13238 22112 31890 31887
31886 32434 32431 32432 32433 32462 32435 14244 22524 22523 22518 22918
16645 33282 10976 17235 17236 15212 23069 23064 32457 32456 32594 22919
22909 15946 16970 17610 16972 16971 15951 16969 15952 28738 32731 17656
17654 17658 17655 17652 17653 17657 32443 23065 23067 29800 32733 32684
11076 11074 32705 11079 11078 31873 11075 11077 31870 32706 32698 32699
31869 32700 31868 18294 18290 18278 18274 18276 18273 18275 18272 18277
18271 18293 18261 18260 18291 18289 18295 18286 18279 18282 18281 18285
18283 18280 18269 18268 18270 18267 23061 18264 18266 18263 23060 23059
18288 18292 18287 18265 32440 32441 19976 19990 19983 19978 19975 19988
19985 19981 19982 19979 19987 19980 19984 19977 19989 19986 23070 32444
20167 30939 30904 18647 18648 18646 18481 18478 18482 18479 18475 18474
18480 18477 10021 10017 10023 10022 10020 10019 10018 10013 10012 10014
10016 10011 10010 10015 10009 20370 20375 20371 20374 20373 20372 20369
20368 18476 31691 32746 32696 32740 32739 32690 23071 23066 32447 32449
32448 23068 23072 32446 32453 32597 32596 32595 32436 32437 32442 32450
32451 32452 32454 32455 32459 32458 32461 32430 32439 32445 32742 32692
32687 32688 32691 11018 12143 31251 31246 12130 12954 19959 19957 19958
19960 665 13171
634: 23618 1224 1754 3565 30468 9444 5488 8262 7348 6868 6622 27429
635: 14764 11553 11554 11555 20378 33057 33056 1778 2708 3774 3531 20303
20376 29086 26906 33244 33246 33240 6321 6274 6149 8313 7727 8312
7468 7008 7839 7007 18862 21913 21911 21909
636: 1280 32021 29146 29147 15401
637: 1395 1444 1624 3898 2354 3779 6488 5833 5539 6543 7920 9031
7541 8075 8676 8650 8651 8077 6637 7921 9030 8076 9198 8425
8827 8508 8074 15858 9549
638: 23303 22414 1045 24942 2002 2221 3463 2088 22493 15331 22912 20078
6408
639: 11325 17887 33493 3437 2452 22003 4611 5279 6243
640: 33196 21137 11447 24851 13573 12197 30266 30278 27171 30106 31637 13127
13131 32899 32927 27851 27837 27843 20047 32969 10282 10318 10258 11198
24093 24078 16153 16134 17378 17401 17387 17317 25768 21064 13401 13395
12648 18329 13940 13867 13894 13923 13897 13922 17664 10545 14199 21031
15705 14583 15200 15157 14988 15001 12889 12883 15556 14126 17530 22468
22431 23797 14460 15778 17022 15508 17844 17829 17775 27880 16265 29829
16256 28218 18786 21194 16706 16694 25998 29831 16871 32372 27581 16809
29858 29889 25981 29899 11907 11929 11925 11965 19219 19258 19249 19239
17117 17129 17114 18608 18091 18024 18043 32941 28543 28591 18700 18707
18727 16392 16314 16333 16327 24728 24746 24761 24731 18946 18895 18977
19072 19132 19136 19121 12812 28910 12789 12059 12048 17968 18218 18171
18165 18156 18210 18180 18125 14059 13990 13999 14057 18484 19418 19383
19414 19305 19836 19856 19867 19840 23448 23580 19486 19566 19612 19694
19570 19754 25943 29920 25001 20980 20396 25592 30136 25029 20597 20770
32213 32189 23201 30346 30332 22330 22347 12287 21012 33432 33415 21628
21660 22565 22567 22237 22286 22979 22858 21413 21416 21470 21445 21469
21468 21406 21442 21319 21381 21322 21378 21317 21248 21241 21274 24220
25919 30489 27172 23966 23940 23919 24007 30190 30181 25173 26086 29999
25103 25848 25096 25819 30062 25084 26588 28266 25132 30049 26950 25224
25230 25680 27658 27714 27784 27751 27779 27789 27740 25443 25465 25319
25330 25428 13024 11683 13026 11682 10715 10703 10683 10662 10698 10671
10690 27090 27065 13752 31071 26413 26497 26506 26405 26226 26203 26211
26283 26330 26219 27982 27213 27222 27232 27230 9508 9506 12029 11532
16481 16462 16465 30623 12416 12380 12419 13323 13317 16189 16226 26135
12527 17913 10402 18823 18820 26020 14927 14895 27462 28205 28183 28181
14311 14310 28034 27329 27338 27341 29306 28351 28149 28468 31834 31831
31848 31825 31830 31846 31847 31845 31842 31839 31828 31840 31822 31821
31820 31819 31829 31832 31827 31843 31841 31838 31818 31837 31836 31835
31824 31817 31844 30440 30439 31833 31826 30442 30434 30425 30420 30437
30428 30427 30426 30424 30422 30421 30418 30417 30441 30438 30436 30435
30433 30432 30431 30429 30419 31823 30423 30430 24796 15368 21686 13704
13720 14309 30732 25503 29697 16418 11095 30378 18768 15441 15627 15380
20143 15235 10219 13493 1489 1216 1728 14308 11605 11601 2020 2469
3616 1876 2467 1952 2468 3857 2901 3615 3170 3060 3870 3871
2620 2008 3810 3172 22913 21950 30714 29348 22021 28956 32634 24042
29210 4679 4826 5739 4640 5286 4271 5740 4638 5741 5700 4911
5743 4824 6381 5742 4912 4643 4752 4749 4259 8026 9078 8048
21167 9261 9262 7626 8049 9256 8410 7851 8796 8757 7028 7505
8798 8797 23057 23058 7506 7843 7200 7964 8411 8807 8207 31115
14307 18425 18424 18423 18422 18421 20322 9807 23338 18814 18815 10554
19421 15636 20646 23667 30383 30379 30377 30380 30488 15066 30409 21130
21127 21129 11426 11423 9858 11427 11424 11290 15064 11422 9861 22382
22549 9857 11425 20275 15062 9859 31375 15063 9860 31110 15065 26821
32151 30385 27526 30381 32150 32140 30386 32146 32143 30388 32154 32153
32147 32145 32144 30384 30382 32149 32148 32142 32152 14241 9862 32155
30387 32141 11408 11407 32002 30874 30872 30876 30875 30873 30867 30869
30871 30868 30870 11385 24924 32156 30408 30399 30414 32157 25523 30407
30415 30416 22949 13251 31112 13250 13248 13246 30412 23693 30410 30411
30413 28675 28725 28747 31849 29809 29804 16581 12463 31185 31191 31222
31180 22495 22496 22494 17280 21524 31113 30909 30908 22914 11029 12107
31264 12105 12103 12970 12957 21489 31114 31560 12173 23305 32257 32256
32255 21126 21122 21124 21123 21119 21121 21120 21125 21128 13199 13186
13160
641: 24477 26752 22776 28635 1333 3266 2912 2833 2777 3125 3823 3822
29040 6131 5278 31892 29743
642: 25337 3496 2722 3586 3169 2921 2646 2138 2810 2136 2022 2166
1957 4027 2922 3453 4382 5726 4903 8804 8029 8030 7124 7384
7858 6552 7527 6652 8677 7553 7554 6831 7660 7886 6621 6945
8881 6944 7469 8204
643: 28796 29240 29122 28987 6580 6860
644: 11446 30701 21802 18014 33328 22063 22387 15483 15484 21096 22528 22383
1617 3483 22384 22385 25023 13050 31238 21897 14294 21781 21782 5764
31928 9083
645: 23500 23585 24550 24396 26815 26814 26656 25655 22730 22714 18252 18871
947 3052 2079 3449 20312 21967 22506 22074 5931 5355 5671 4825
6168 6542 5703 4296 9212 9153 9412 8314 31368 24948 20658 18863
14282 18249 15449
646: 23549 24517 26669 22748 19193 20919 15381 15446 17879 27148 1760 1095
1511 1208 1805 1743 2694 3648 2205 3207 2453 2695 3145 2693
3146 2667 2280 2840 2474 9578 29053 15743 6048 5880 4406 5592
5450 6047 6113 5825 5503 32109 20618 15623
647: 29227 20848 27628 27627 27629 12756 21107 14863 28236 31628 29942 17359
17376 10883 10904 13932 14522 14542 14480 14479 14487 15146 15039 12918
12869 19002 19016 17502 17543 23811 17854 17807 16782 27597 11937 21859
21887 21888 16942 17116 28537 28536 16379 24730 18947 12842 12799 18162
18161 18221 13977 19839 23627 19565 25963 25962 26694 21718 21717 12471
12472 22366 21465 21401 21372 21298 21296 21332 21218 21253 24044 23995
25181 25865 25854 25116 30088 25692 27713 27676 26439 26206 26386 27956
27229 9498 16478 26145 16174 16048 14905 14906 27368 28142 28143 23279
18413 13691 10555 11134 29722 29712 20204 12485 28750 31229 30920 18994
12134 12973 12175 29366 15665 30686
648: 23288 33200 13579 32932 27838 13966 10317 10314 10313 29581 16162 17400
17386 10907 13379 13425 13418 12645 18347 14545 14360 15722 15668 14505
14511 14481 15136 14977 12868 32295 32291 32623 19011 15550 14147 14144
14162 22455 22454 15278 23788 14459 15816 17032 30980 17037 15618 17841
17838 17851 17821 27895 28073 16266 16286 16783 16853 16867 27542 16838
11993 11926 19259 19269 21882 17172 17455 17099 17104 18610 18594 12744
18050 28621 28523 18729 18693 16329 16350 24687 24706 24704 24762 18970
19056 12779 12820 9872 9871 18211 14042 14041 19382 19415 19789 19807
19805 19868 19664 19606 19572 25940 25944 25959 29919 24447 20996 21715
12476 23191 12339 22374 12289 24307 33398 21654 22849 21365 21251 24218
24217 24219 27173 27177 25738 25740 25790 25863 30061 25111 28836 26976
26969 25697 27671 27707 27737 11675 13036 24967 27107 27116 13776 26498
26486 26262 26391 26246 27981 27257 27217 27282 27268 9460 9494 11516
11518 16470 12403 13305 13344 13338 26100 26159 10373 10369 30520 14902
28175 28180 28401 28374 28487 23344 32383 13666 29709 27467 3445 3476
2400 2068 2768 2532 2262 2292 2122 2624 30587 6484 6526 6422
4359 6063 4820 7449 7662 6846 7376 6711 11064 17981 19182 19179
33011 33010 28709 12456 31162 31291 20873 21090 14518 12991 12989 28250
10622 21772 16602 12183 26860 23084 13205
649: 1325 1453 1322 2614 3430 1946 2397 3474 2235 20211 29084 28941
28942 21989 29194 28940 29005 5623 4871 6510 6509 6448 6106 6882
7805 15435 27456
650: 17227 19279 14726 15225 12211 30241 13104 32413 13086 26572 32915 31938
33446 14399 14398 31334 31333 31332 30461 16960 22190 27463 14725 16111
27861 24079 16157 17327 10882 12550 24228 25148 21084 13434 12642 12686
29271 18346 13884 16763 9798 21030 18548 14521 14361 14588 9725 14512
15197 15036 12882 33253 15230 32612 15118 19001 15542 17510 22457 23785
15765 30959 15521 17772 28057 16271 28095 18788 21189 16704 32357 27593
16840 29842 11974 18444 21547 21878 17208 17443 17118 18621 18048 19427
28533 18699 16313 24685 18904 19058 15239 12050 9882 17967 18196 13991
18496 19366 19786 23519 23617 19477 19622 19755 25942 25960 20020 24497
24557 20333 20979 17980 15224 26732 26670 26788 25572 25652 30119 25888
22678 22718 20790 32223 23225 30343 22367 10086 12310 24305 21015 33382
21644 22191 22197 22192 22193 22194 22195 22196 22198 22199 22200 23369
23367 22243 22845 21435 21352 24180 23261 29475 24063 24006 25724 26077
23167 25070 26599 24284 25126 26984 11450 30143 25488 10663 27058 25298
27153 31078 26407 26265 26190 9451 12031 11525 29774 30643 12413 13353
16194 16192 12524 24586 17925 10368 30521 16055 26042 12607 12027 18639
14934 10594 28206 27366 28486 13706 29720 9669 16927 20261 1086 31581
31582 13243 11583 3901 3213 1903 3902 3900 3038 3124 3214 2120
1895 32024 33079 5174 6154 4436 4435 5431 5542 5430 5175 5322
7961 8232 8474 8706 7504 9264 7190 8707 7680 8231 7712 8977
8940 7542 9393 9388 8127 7838 23889 23890 11319 11320 10862 21924
10859 10860 18414 9757 9760 9759 9758 17986 16024 16025 17987 16022
16023 9756 10861 20155 10858 10501 21159 24952 10500 11173 31902 29598
13460 11229 20702 20441 32985 13798 9439 9440 22189 23101 23103 23102
30877 30880 20591 30878 30881 29409 29408 30879 28702 32125 16498 29795
12450 31583 31147 31306 20872 23363 23356 23368 23364 15664 10467 10466
19919 29413 20828 20827 19918 23361 23366 23362 23358 23371 23370 23891
11318 23365 23360 23359 23357 30932 24147 12102 16594 12171 29153 26859
22215
651: 33238 27449 13136 16094 33354 12552 33264 25772 25751 25153 31231 29218
10632 18573 20141 27904 28221 18115 23593 19592 20423 20392 32217 22301
23250 23132 23978 23989 31035 27166 31671 31672 25357 28341 9993 1053
852 1360 1675 2940 2558 2559 18453 18452 28973 29259 29136 9660
9661 9658 10642 9657 9659 9655 10640 10645 9656 10641 10643 9654
10644 24944 17275 32401 31348 28856 27128 5619 4980 9241 9060 18764
32408 33509 17281 20539 20538 18505 18400 16546 16497 16575 16574 29806
12097 12142 12091 12087 26528 20129 14791 24633 33609 32843
652: 33467 1564 1241 899 3638 3637 2158 2925 3438 20366 10577 24010
21545 5572 5034 5195 5075 5771 4992 7267 7578 6805 8117 7429
24809 652 15819 15437
653: 1406 1319 1284 1424 1485 1383 1320 2430 2303 3636 3349 2988
29047 21951 30715 10053 6350 4600 5508 5375 4163 6240 4497 4594
5196 5067 6077 5376 6299 5276 9149 9148 9151 9150 8011 7507
9218 6925 7277 7647 7648 9015 7209 15464 17212 15890
654: 1466 1796 3407 3642 3369 2389 9580 32708 8986 7387 9396 31355
32425
655: 929 2958 3519 1911 6295 7562 7127 9029 28672 20448 20053 20054
656: 5365 6120 4161 8982 7071 7072 8152 6737 6619
657: 23604 24431 24474 26625 25602 1001 1119 5624 5419 24788 22941 17258
658: 9583 9827 33632 15221 12209 30232 13146 12216 32923 20326 33012 19276
18875 14416 12995 10507 27828 16021 12540 16013 15928 15220 10301 30303
29577 27869 29952 16158 16140 29584 31027 17329 12563 24226 25757 21046
12662 12702 18314 11711 13865 15222 14192 21036 18551 14348 15678 17722
14595 14685 14476 15167 15011 12893 29594 32631 15124 19031 29220 15527
15561 17871 17519 22484 31670 23790 15780 17019 15519 27898 18796 21184
26000 27594 16822 16890 11973 21865 32570 17199 17459 17137 18593 18074
28590 18849 10510 18691 18716 16372 31669 33617 18905 18968 10508 19096
12829 12058 9893 17965 18189 13994 18494 19404 19350 19884 23716 23533
19501 19599 19764 20485 20458 24349 9697 20340 20975 20401 32990 20556
10505 25603 25893 22802 21726 20816 23211 12336 30347 30329 22318 10090
12296 24304 21017 33404 21647 22255 22852 21371 24197 24199 24203 23255
23271 29476 25932 23922 23979 25719 26080 25798 25866 23169 27501 25855
26582 24257 28281 30044 28841 26979 25700 27731 25486 10666 24963 27111
25284 27152 31053 26402 26224 26187 27254 11382 9524 12044 11477 16473
16479 30595 12381 12369 13347 16215 12499 24575 17910 13274 10409 30530
16045 12621 12247 14919 10838 10592 28160 27356 18001 28134 28452 23684
23686 23687 22184 10506 21106 31738 22498 26888 33287 33288 19703 13675
14117 12331 12333 12323 12324 12325 12326 12327 12330 9593 16428 29730
29715 29733 29706 9755 33289 21163 20259 1712 21853 1673 1240 1745
1049 23685 21852 25016 23741 11587 3218 2080 24245 2809 2265 2300
2889 1836 3697 2890 32654 31668 33351 33352 20964 33150 30659 20284
18473 9946 12320 12322 12321 22166 12318 33117 6040 6261 4773 4618
4751 5869 5868 4769 5867 5966 5870 4113 4403 6164 6073 4303
5204 4984 4975 6380 6265 8122 9340 8489 8759 8760 6814 8761
7353 7098 7278 8762 7246 9155 9154 9414 7040 7120 15485 31361
16722 23688 22527 21529 28110 19770 14947 16515 9992 19270 11473 23091
23090 14759 9720 11166 33131 16635 15070 21703 11090 11057 23660 20626
22971 12328 20186 27451 11324 20045 12329 11271 13478 30848 11421 28741
16550 12443 17699 29338 23691 31205 31330 31319 20885 20881 20046 23690
30943 31608 32005 12332 12106 21763 16588 12165 15922 26834 10509 23682
23683 12319 23689
659: 1583 897 864 1752 1753 3879 2447 2944 2347 2035 1855 2831
2449 3405 2448 2348 2987 2446 2102 3005 1837 28851 5314 5315
5041 4520 6487 5885 6398 5762 9263 8967 7903 7397 7625 8463
8394 8592 7498 8591 7491 7107 7108 6694 7902 9403 9972
660: 23609 1740 1167 3966 17220 29141 10962 10959 10961 10960 5469 5115
5095 5679 6162 4612 4702 5678 6330 7833 6885 8478 7145 8483
6975 9065 8602 7144 32839 15455
661: 32709 20936 10316 10255 10288 10315 11181 17350 14107 13388 18353 18301
21034 15713 17068 14122 22433 22432 22429 30981 17011 19931 17840 17808
17839 17853 27556 27573 29893 29901 11918 11928 12008 19260 19213 19261
17477 12741 18023 18026 18025 18021 28607 28546 24701 24779 18984 18939
18951 19085 19130 19129 19128 19291 19352 19802 19886 19747 25936 25970
24494 20990 22370 22313 33416 33424 33426 21593 22880 22838 25745 25793
24662 26590 26944 27069 27040 25304 27267 27291 12042 12377 10397 10393
10394 12598 12020 18634 28355 28417 28435 28429 20193 33303 1236 3768
3025 3594 3222 29117 28969 5669 4762 5719 5851 6326 4529 5862
6300 4816 6485 5035 6423 7482 9200 8897 8012 26091 8896 7123
6884 7044 9415 9070 6642 6750 19181 21916 19180 27126 28871 24144
12990 31452 11760
662: 1184 2069 2761 3420 2710 28965 6285 5432 5433 5522 6160 9041
7381 8778 8198 6582 32890
663: 27643 10279 17746 30970 17798 19223 29217 9705 20415 23213 23986 27499
25120 28269 25696 27811 25484 13760 26432 26357 31386 12502 12846 10847
21138 31009 31011 21851 29682 1267 1411 22125 20313 28971 4418 6421
8943 6921 7925 8433 31457 26824 32647 28734 12151 31265 13174 32847
664: 27204 13545 13557 13540 32267 32239 25069 25802 13130 13098 32917 16913
11238 23100 12539 23410 10291 27855 9435 29376 11213 25899 17308 17328
12585 24237 25776 25147 13368 16306 19741 18254 18356 11713 13871 14204
21032 14547 15691 14596 22092 15175 12884 31383 29373 33570 21577 32599
15122 15544 17516 17521 22424 22471 15277 15809 15786 30972 15620 17810
16255 16705 16868 32375 16884 29866 11992 19209 21857 17126 12730 17999
18097 28578 18678 16368 16317 24737 24759 18976 19059 12787 12081 12055
9880 18181 19343 19847 19865 23529 19615 19749 25947 20454 24360 20402
20575 20549 26708 25051 25031 25037 22712 20789 20782 32227 12356 30369
22353 33395 21595 22292 22281 22853 21432 21349 21273 23246 24043 29969
28256 27188 25731 26083 24650 24659 25822 25082 26591 24253 28263 26928
25222 27669 27757 25490 10677 24981 27092 27032 31086 26412 26221 26183
27933 27979 27262 27231 9478 9472 12040 11503 13294 26104 12519 10363
27994 27388 28383 28123 28495 28324 21103 15365 13685 14792 22886 29700
16429 9671 25382 14882 32412 32963 32964 23118 18644 32472 9736 17255
18378 27140 18432 31706 31705 16649 9436 23739 23738 12430 33514 29181
15477 31665 30757 13524 25026 16559 29799 31175 13048 30947 31600 22185
14115 14116 33005 11019 12976 21753 21493 12172 12174 26843 26855 31443
665: 30490 9787 13546 32703 32704 32702 31872 31904 27618 32743 32693 32738
32741 32689 32745 32695 32747 32697 11055 14342 14340 30991 29471 29462
29474 29473 29463 29469 29464 29468 29465 29461 29472 29467 29470 29466
21565 21561 21566 21564 21562 21563 21567 17222 633 13102 15761 555
31493 16973 15947 19951 11292 10253 33349 17330 31287 13877 13929 12852
13945 22533 20213 19777 19775 13005 19778 15706 15198 15201 14990 14987
12919 12924 12914 14798 17503 22453 23776 17770 19950 19943 19941 19949
19948 19945 19942 19946 19947 19944 27918 16777 33613 25983 11972 17159
31491 31492 17119 17155 19971 19973 19962 19970 19966 19969 19964 19967
19965 19961 19968 19974 19963 19972 19952 19956 19955 19953 19954 12726
18051 28569 18737 16384 18906 19111 18213 18231 14013 14047 23714 23502
19779 19604 19649 19670 19681 29928 20011 20009 22976 17869 17868 26770
25660 25049 22729 22373 21598 22296 21392 23160 29477 23917 23987 23992
24642 29994 25815 25109 25839 24259 30155 27697 27767 25473 11663 13042
10675 13738 31059 26427 26263 9493 12523 16075 12259 28187 28032 15366
15364 22522 32729 22515 22516 22517 32734 32736 31683 17946 32877 28331
33498 33502 14271 32827 14071 33530 32828 13661 32393 26162 10162 10156
10161 10155 10157 10160 10159 10163 10158 10154 31751 31750 32876 20185
28330 17954 17950 17951 17956 17953 17949 20158 13015 31753 31752 31748
31749 31755 31754 31756 31766 31765 31763 31764 31757 31758 31762 31761
31760 31759 29268 31494 32513 31490 16427 21001 17234 17239 15949 17948
17955 22090 11832 11831 10477 32956 32957 32875 32874 17952 17947 33503
33504 22098 23913 23911 23907 23905 23906 23904 23902 23901 23903 33499
33500 11835 11833 11841 11834 11839 11842 11845 11846 11848 11847 11840
11836 11851 11843 11838 11844 11849 11850 11837 32878 17945 33501 20159
33497 16931 21576 21575 21574 9853 21211 11449 10441 18651 17659 14445
14443 10440 32460 15451 14444 14805 15406 15731 9441 24636 29333 24595
13736 28860 14270 15474 14281 15434 15730 32844 32502 20140 20139 9545
15413 1787 1767 874 32438 23074 14292 33281 11584 9797 11630 32871
3988 3280 3989 2729 2716 3284 1905 15927 2918 3885 1938 2324
3643 3447 3670 23334 2222 2623 2295 3821 3111 2239 4075 3625
2189 2044 3502 24853 2237 3446 3950 2426 3884 2540 3344 3504
4026 2939 2071 2019 3990 2571 2590 3194 3680 2980 3805 2188
2602 17237 17240 31489 20222 15630 31889 31888 22143 19277 13282 14769
28995 28963 22004 21968 28964 21969 32680 20282 29110 21902 32728 32682
17629 32701 31871 16965 16966 15945 11080 11070 16964 16968 16967 15950
17609 11081 11082 11084 11073 11071 11083 11085 11072 11086 11088 16974
15948 33115 9730 33116 31877 27442 19468 19467 9731 9840 9591 31885
21569 21573 21572 21570 21571 21568 14824 14339 23073 21900 21899 21898
21901 32726 32681 32635 13479 21903 32737 32686 32744 32694 32735 22526
22525 22520 32685 22519 22521 32732 32683 32727 32730 10979 10978 29628
29629 29633 29632 29630 29631 32244 21680 5819 6431 5729 6067 5555
5421 5918 5921 4799 30397 5920 4798 5203 4797 5422 5424 4796
5917 6288 5919 4208 5058 6175 4795 5904 4244 5647 5906 5849
5554 5423 6176 5556 5443 5053 5818 4581 6194 5014 6177 33348
10194 8574 9266 7909 6719 9267 6707 7875 9331 7704 23005 9265
6721 13446 9330 6639 9332 8526 7708 30192 7153 8942 9333 7216
9011 9133 8216 8217 32533 7812 30662 8604 30661 6626 7754 7604
9232 7373 7834 8941 8724 7602 7603 7606 7600 7601 8129 7067
7179 7607 8218 8527 7887 7817 7645 9139 7266 8324 7099 7605
8538 7259 11658 11115 10197 10195 10198 10196 17241 31014 21140 11133
13008 13009 19362 13014 20171 31387 33496 32580 13017 20165 20166 20164
20160 20161 20162 13016 20163 20169 20170 20168 16654 20172 20173 20175
20174 20176 20180 20177 20178 20179 20181 20184 20182 20183 13778 13779
21537 22749 30727 13733 13238 22112 31890 31887 31886 32434 32431 32432
32433 32462 32435 14244 22523 22524 22518 22918 16645 33282 10976 17235
17236 15212 23069 23064 32457 32456 32594 22919 22909 16970 15946 16972
16971 17610 15951 16969 15952 28738 28718 28723 32731 17655 17658 17652
17656 17654 17653 17657 32443 23065 23067 29800 32733 32684 11076 32705
11079 11077 11078 11074 31873 11075 32706 31870 32698 32699 32700 31869
31868 18294 18290 18274 18276 18273 18272 18275 18278 18277 18271 18293
18291 18289 18295 18286 18283 18282 18280 18285 18281 18279 18263 18269
18267 23059 18270 18288 18292 18287 32441 32440 19976 19978 19983 19981
19975 19988 19985 19979 19982 19990 19987 19980 19977 19984 19989 19986
23070 32444 20167 30939 30904 18647 18648 18646 10021 18474 18479 18478
10023 10022 10020 18482 18480 18475 10017 10019 10018 18481 18477 10012
10013 10014 10011 10016 10010 10015 10009 20370 20375 20371 20374 20373
20372 20369 20368 31691 18476 32746 32696 32739 32740 32690 23071 23066
32447 32449 32448 23068 23072 32446 32453 32597 32596 32595 32437 32436
32442 32450 32451 32452 32454 32455 32459 32458 32461 32430 32439 32445
32742 32692 32687 32688 32691 11018 31251 12143 31246 12130 12954 19958
19957 19959 19960 13171
666: 851 1228 3409 2227 4081 2501 4041 28997 29149 31495 30655 29013
20283 29012 31106 4203 5590 6209 5016 5043 7560 8869 7441 6883
6880 7238 9369 6987 7551 6932 7530 8892 6821 6847 8086 27447
667: 14844 20931 9597 27612 30239 30221 30248 33055 32903 32926 33571 14811
11294 10268 10251 24082 29935 29948 24073 12594 17422 17351 17368 25136
24857 11725 13920 14211 31882 24148 20214 19774 19776 10936 15957 14680
15199 14989 12931 28230 14157 17508 22466 30965 17041 19927 17688 17768
27900 28079 16800 16797 32349 32373 29876 20515 29878 12010 11889 11986
12014 17138 17244 28551 15238 16386 24752 24736 24724 18877 18942 18888
19076 12796 33624 712 12054 18214 18167 14022 19859 19845 23586 19595
25000 9694 22975 17979 26706 26707 20937 25586 25588 23737 20801 23192
12357 22299 24297 33391 21623 21608 21444 21321 21345 21398 21265 23253
23268 27189 26063 27477 25818 25092 30145 27672 27683 27805 27725 27800
27787 27746 25464 13033 13023 10697 24980 13756 26509 26501 26448 26517
26228 26341 26269 26310 26282 26372 26257 26300 26255 26253 26301 26387
26273 26321 27284 27261 9458 11542 10400 16049 16065 12603 10973 12220
10823 27018 32757 17860 28010 27992 27999 27383 28368 32384 27202 17247
20088 17591 15397 15367 26887 15744 15760 22183 14391 20837 10647 27165
11410 13701 33357 23721 20935 9740 22202 16438 14852 2249 3844 2381
2949 2773 3987 3704 3700 3835 2084 3761 2630 3767 3895 2737
22150 22140 22155 22156 22046 22045 28978 11639 11436 29341 6212 5284
5061 14564 7316 7627 14563 7325 14562 14561 7481 9355 7185 7865
9038 9294 7761 8000 9184 8429 8934 7759 8672 7783 9401 10856
17043 10608 33610 32117 21481 21208 33304 26886 11304 13239 17568 19280
9596 23829 11809 12996 12462 24921 31141 31163 17902 29212 24146 22203
24939 11037 12094 12090 12138 12104 12952 28247 10605 32972 571 24925
32037 28047
668: 20378 1045 24942 3891 22493 21904 21905 20582 20581 20583 33240 33242
33239 11556 20078 20254 15374 29207 10482 10480 18862 9804 11769 33268
29330
669: 22091 14386 14387 10582 1773 1288 1497 1358 3741 2473 2440 3968
2121 3685 1830 21977 14553 12487 5767 9147 6967 7814 8507 8849
6933 19939 14744
670: 15939 33485 1043 1334 1737 3773 2852 3399 3486 3497 2610 3696
2564 2611 3428 2677 2291 3269 3362 3413 2785 2959 2853 3270
3717 3376 1978 2628 1915 2099 3532 3366 3652 2153 2050 2042
2332 2244 2696 2209 2960 2025 2152 22001 30710 21945 14405 5081
6430 4982 4735 5839 4444 4969 4983 4821 5912 4988 4394 5636
6517 5540 5806 5420 5559 4853 6086 5809 6163 6455 4811 4539
6304 4989 5541 6034 6193 6171 5988 6039 5593 4546 4568 4589
4305 5087 5202 9051 7701 9152 8562 7341 7922 9161 7797 8052
8230 6661 8479 8893 7319 8979 7503 8559 9410 6608 7125 7693
6554 7672 7416 7342 8267 7256 6631 6611 7073 6924 6653 7885
6895 7066 7171 7198 7788 6854 7371 8113 8114 7172 6550 7042
7419 7183 7203 6806 7065 6956 8370 7032 8138 6877 8652 7074
6859 6607 7169 7192 7141 7191 7075 7363 8369
671: 11774 1641
672: 23439 24348 24530 26646 26778 25573 25637 22632 22694 11302 32833 15444
985 1422 1347 1929 2162 2218 4085 2197 29243 4557 4284 6228
4587 4641 32713 16955 11122 30717 24790 20703
673: 30014 1777 1647 1646 1645 1213 1174 1582 1449 3406 3271 2797
3033 3034 2502 2516 2799 2798 3192 1991 2048 2829 2495 4031
29137 4774 4678 5913 4537 4685 4591 5066 6100 5065 5101 4377
6200 4809 5794 5261 7564 7710 9305 8197 8795 8343 8572 6605
7722 8603 9024 9371 8196 6687 27448
674: 25752 14973 28219 909 975 29246 24797 6318 5696 20049 7876 6878
15452 15443 774
675: 14764 31003 31002 22414 29460 895 1101 1047 1441 1592 10878 32955
10879 13842 13843 26616 26611 13225 13220 26619 13221 13222 26613 26617
13224 26618 26612 13223 26610 26615 13226 26614 3693 3518 3316 3195
2651 2250 2657 2848 3619 3317 1917 2734 1831 1893 3308 31128
15331 5550 8210 8816 7017 7399 7433 6962 7117 9424 6676 6833
24614 32849
676: 14847 33198 13532 20846 27626 30225 15868 17222 13147 15761 26911 555
33574 14643 14650 14649 14648 14647 14646 14661 14659 14660 14642 14664
11417 21673 18297 14838 10252 10325 30305 30299 29569 16125 11188 17345
17375 10881 12562 12574 12575 12584 33214 9868 31287 18310 13885 11712
13868 11717 12854 13947 13855 10646 22533 23036 20213 18550 18568 14520
15706 14581 15132 14971 21583 17242 32629 19000 15545 17497 17504 17494
22453 23777 23773 23825 30986 15612 17769 17857 27918 16699 32367 32364
27567 15894 29908 20514 11948 12016 18440 17159 23412 17441 17155 12726
18051 28600 28579 16332 24727 18934 18920 19111 12813 18200 18208 18175
18164 18145 14011 19402 19835 23714 23525 23548 23554 23479 23573 23461
23419 19779 19593 19556 19544 20010 24544 24345 24521 24339 24420 24534
24484 24372 24382 24388 24426 24564 24460 24548 24536 17988 20565 22981
26767 26808 26805 26750 25564 25598 25625 25562 25589 25626 25886 22660
22661 22784 22697 22685 20810 23228 23183 30352 30365 22373 21598 22568
22275 22293 22270 22876 21392 21270 29477 23656 23977 23945 23941 29994
27502 25079 25831 26594 24260 30030 30152 27711 27730 27778 27815 25458
13032 13763 13748 31059 26513 26399 26466 26472 26446 26204 26354 27226
11379 9457 30677 13303 12522 12526 17921 22546 22545 22547 16079 27423
27995 27330 28392 28447 23088 18519 18515 27149 13662 13719 13689 13652
32393 13488 30749 26162 14639 14644 14645 14667 15259 14446 19771 24813
29727 16407 16398 11818 29265 11227 33606 18992 24125 1520 1618 1795
989 10351 32429 10352 10350 32428 27464 11625 32871 1982 20222 33459
22143 13282 20291 32173 9595 19468 19467 22415 23073 21903 21162 6537
5227 5936 4378 7439 7177 31014 11276 32580 13017 22749 30727 13733
13238 33358 22112 16301 15343 15054 29427 29428 22538 22537 22540 22539
33516 14401 29737 15821 15820 22542 22544 32811 22543 22541 31501 31500
32462 32435 31912 14295 20634 20619 20689 33140 20714 22945 22536 22534
22535 31563 31453 22956 24988 24989 24990 18527 13240 32880 11405 15212
14465 25195 22909 28778 28766 29792 14638 14652 14651 14653 14654 31140
30939 31998 14663 32446 25194 25193 25192 32455 32430 32439 11030 12122
24326 12953 22999 16600 12181 12191 24926 14641 14640 14662 665 14655
14656 14658 14657 28782 14666 14665 9586 13210
677: 23619 24341 22768 1094 902 2806 2150 3976 2805 31468 22024 4651
4269 4664 7972 9130 17661 14862 24783 20130 9558
678: 22815 10867 17570 20799 10084 29459 1517 10511 2884 3580 3113 29008
33523 24940 4273 9088 15942
679: 23441 24518 25633 22769 1827 1969 3039 2031 2318 3607 3419 3514
4060 3418 10604 29352 22007 21943 30658 6283 6169 5877 4562 5007
4955 5372 5814 4662 6875 6770 7186 9423 6865 6920 7229 9253
9374
680: 17547 26131 11137 1077 1801 1087 1025 995 3752 3623 5660 4707
5209 4551 8767
681: 18507 15657 11386 32182 27460 33120 22123 21535 17997 23446 24523 24478
26695 25631 30765 22780 22794 23694 28912 33137 27441 18528 29269 33254
12423 13789 1067 1474 1461 17998 3694 2825 3729 3358 2424 14252
24829 15068 33092 10359 21992 29133 9998 18100 15615 10542 31655 10543
13615 17695 30446 18405 6386 7899 8987 8025 7960 8276 16029 17647
17646 10572 31657 19925 19523 32118 18541 19146 20678 20961 24640 29454
10967 10332 22562 30763 30761 14634
682: 22617 11553 11554 11555 31546 14396 20378 24942 14949 4087 2672 21773
11103 11105 11106 11107 11108 11104 29299 29114 19429 21906 21905 20580
20582 21904 20583 20581 33240 33239 33242 33241 13085 11556 11557 20078
5756 4193 4438 7584 11551 10481 10480 10483 10482 18862 21913 21909
21911 21910 21914 21907 21908 22620 22619
683: 904 1821 1139 965 1465 919 3837 3839 3957 3956 3287 2040
1948 3836 3589 3120 29082 30709 30023 29401 30708 30021 30022 4950
5962 6182 4748 4747 6511 4746 6725 9193 7148 9192 8571 8202
6811 6601 26995
684: 27516 1562 1373 1663 2010 3864 3171 2009 2011 3865 2885 2596
2201 2211 2039 3065 22010 4660 5253 6470 4595 6496 5538 4153
4804 4147 4805 6123 4279 7801 8364 7804 7264 9271 9430 8815
8365 8661 8742 7253 6936 7937 6985 8758 9004 6584 8898 9002
9003 8823 8570 20065 12483
685: 23616 24472 26630 25629 22789 884 1252 1301 2740 3811 2533 3200
3110 3672 3673 2787 2778 2786 10496 30211 5879 6395 4380 7904
7894 8160 9135 8623 6562 8624 6747 7893 8649 7936 7726 7391
7403 11702 23176 25364
686: 1241 1438 3438 3417 1872 2276 28894 28929 5572 7939 8151 24809
32842 32465 9559
687: 15209 15048 19335 24434 26655 25595 22656 20761 9722 28883 18446 1112
20042 3542 3101 2374 2414 32391 5687 5335 6547 7861 9126 8674
8675 7713 8947 7050 7862 32589 32859
688: 1306 1278 2154 3397 28937 4157 5753 6049 5337 4570 8164 9074
9075 8909
689: 18507 27460 33120 26626 11788 28912 27441 21213 33254 13789 1643 1622
14470 22118 11236 24829 15068 10356 15758 9998 20225 10542 31655 13615
32266 32265 30446 5492 16029 17647 17646 10572 30393 9052 8565 6759
690: 15418 960 3969 2298 2327 2364 31469 22025 4348 5891 6074 4778
4143 4588 4776 4777 8400 7039 8398 8399 9288
691: 10215 10214 10213 31712 31713 10212 23494 24543 26802 26798 25618 22720
10947 10948 1352 2542 3041 30544 10131 18874 19708 10953 4116 6055
5893 6788 9072 6651 8517 6919 16983 14434 22940 15433 20123
692: 19354 14329 1598 30203 20307 29023 20289 20288 29125 20290 14245 6015
693: 14857 30541 20063 1024 1162 943 3216 2565 3838 3224 6396 5018
5017 4482 5030 6397 6393 4579 7582 7579 9167 7581 7188 8903
8902 7580 8421 9013 15409
694: 1661 1566 3738 5658 5439 4245 5440 6206 6778 8132 8139 31979
13729 33263
695: 1012 2916 1857 2433 1968 2268 2466 1834 4548 5672 5357 5358
4722 4721 5989 8785 7569 8976 8274 8093 6591 9306 8740 7570
9307 8367 8513 7928 9381 6727 6636 9367 7365 9382
696: 14765 22833 18807 1311 3068 3290 3441 16764 4104 7736 31279 15465
697: 27006 29287 20089 22388 31651 31547 27145 13151 32543 23551 23420 24499
24505 26651 26780 26627 25634 25571 25576 25539 22778 22707 22648 22649
22702 22646 20324 12592 23391 30831 30832 18520 918 1309 25496 30453
29188 33204 21135 21134 25387 11153 29284 29070 15609 21934 22051 11640
32008 22827 28243 33350 28876 11053 13258 11286 9812 27948 27947 30697
9958 32082 25348 25347 32528 17575 15370 10185 10186 10187 31538 10926
29179 6353 4695 5026 4693 6020 5546 5685 5841 5377 4129 4173
6473 5698 5633 6358 4692 5415 4856 4961 6471 4690 5499 4691
4361 4837 6472 6535 4429 5632 4389 4872 5840 4465 5384 5445
5446 6531 5887 4694 6245 4134 6019 6379 5039 5530 4619 5414
6481 6023 4908 6352 5566 5830 4306 8050 17638 14243 17719 24842
19514 9937 28660 28869 22113 16480 13489 13474 20893 20894 11438 11437
14170 11222 9748 10870 9938 15626 29599 13517 15334 15333 15332 19144
11419 13780 18797 32544 16088 9843 33065 17935 22182 20152 13963 23658
9592 22933 20648 13459 16947 32988 19720 33235 30693 9936 11409 13490
18522 14948 14820 12591 14440 11431 31462 19907 31464 27414 31463 14297
28329 17891 11282 32679 32678 28506 9923 14289 14426 22111 22110 30450
27002
698: 1729 1282 5707 5808 6267 4516 5946 5792 31987 21584
699: 24764 12806 19870 25184 25737 27270 27258 11513 15881 1692 1033 860
992 29203 28944 5638 4813 6130 5944 8293 8224 7258 31212 3588
2464
700: 33176 17934 11114 18253 19351 24422 26717 25527 22692 22222 26595 25445
10759 25012 25257 25256 18439 19727 28637 28636 18659 13241 27013 31633
32995 24946 15959 1115 15643 9786 22096 32390 25519 25518 13051 13052
13053 11608 32240 17278 20436 3816 3364 1989 18510 17747 28822 28858
22830 17660 30376 29062 13727 32824 29389 13482 13957 13958 33552 30455
32994 14299 4207 5996 8453 15973 31357 31356 25360 22213 32124 20682
11359 32045 18398 18395 30826 16976 15057 15056 15055 10245 14314 32049
20843 30550 31798 27000 14809
701: 22416 23604 24431 24474 26625 25602 25565 1001 1119 3048 1909 2317
1907 1906 1923 3609 2726 3304 3305 2098 5624 5419 8952 6822
7245 9395 8065 24788 22941 17258
702: 1200 1735 1800 4068
703: 1005 893 3608 2296 1879 1900 22028 28867 30707 7892 6946 11775
15388
704: 10650 23422 24510 26809 26758 22688 30990 30988 30989 19734 1823 3573
2983 2979 3468 2572 3082 2260 2990 3003 3251 2576 3431 2986
2049 2217 2113 1941 2091 2585 4098 1940 2195 17288 6033 6415
5426 4555 6268 4290 5191 6312 5500 5456 7558 7412 7957 8725
6818 9343 7288 7949 6573 7000 6819 8220 7681 7618 8594 7528
7049 7094 6887 9380 6969 8042 6900 7529 7526 6948 27139 17249
10485 30716 20674 19153
705: 23579 24427 24444 26710 22751 20154 32846 1747 1331 3728 29002 5080
4267 4141 4283 8237 6901 8331 8780 9063 8460 8584 13464 20698
706: 32163 32538 30454 12559 25761 21065 13914 18576 15192 15031 14799 18129
23302 25105 30149 27783 25478 10708 26256 29748 17666 9911 16034 15589
23339 15970 15625 9955 1799 11432 11627 4024 1864 2413 4023 13011
25955 13453 15263 30774 16980 32018 11274 13807 18098 10578 33207 4200
5845 5846 4199 4958 7869 7069 8437 7954 7317 9341 9358 22624
27453 27452 24844 11260
707: 13553 13570 23325 23313 9952 23316 33627 33628 824 586 756 787
33621 792 628 613 720 570 567 31789 31790 31787 31135 17283
22061 10307 27854 26917 25763 25758 30001 14356 15528 22478 23030 15424
15425 17023 19935 17830 18079 18081 28521 19090 19088 33615 12060 20971
30118 30126 12470 23207 23231 33374 33376 21658 27497 31788 28277 30038
31038 27957 27972 30627 16171 30524 27371 33180 28669 28668 17680 29309
30013 30015 30729 31392 14451 11816 13265 13264 31578 17603 10474 33620
14277 10539 17720 31984 16625 9971 24627 19515 13000 13001 14795 33330
1305 1704 1102 871 907 906 905 1403 908 872 1825 1398
1402 1126 1545 1401 1153 1456 1251 1544 1376 1414 1527 1326
1382 1354 1494 1399 1370 1361 1341 1476 1587 1504 1817 1623
1264 1639 1417 1653 1543 1651 1590 862 1233 1377 1715 1400
33052 10540 23323 23063 2029 3984 2839 2586 3293 2210 2487 2832
2811 2488 3983 2477 2641 3109 3144 3386 2538 3273 2964 9770
2014 2319 2635 3299 2144 2961 2963 2013 2015 2898 10538 2993
1853 2061 2105 9769 3272 2485 2416 2214 2718 2135 2962 2142
2617 2139 2676 2490 2257 2965 2543 2966 2289 2277 3328 2134
2143 1928 2724 1939 2130 2491 2967 1965 2140 2308 2313 2310
2128 2193 1998 1850 2480 2131 1912 3689 2192 1851 2592 2241
2212 4052 1849 2316 2245 1996 4048 2997 2968 2118 3086 2133
1839 2129 2297 2486 2176 4047 1894 2479 2172 2076 2727 1947
4046 2208 4050 3066 2141 1892 1843 3294 1913 2242 2179 1937
2655 2251 2290 2749 2132 3023 4032 2373 4069 1896 2492 2075
2279 2299 2171 2577 2320 2200 2155 2258 2180 4056 2174 1891
1966 4071 4057 1865 4037 4033 1842 2000 2004 3414 3073 1863
4049 2191 1899 2779 1943 2712 9733 32132 23108 20083 18469 14945
10076 13455 19522 32011 32013 32012 16981 20107 20325 23293 13487 23019
14473 13644 19901 13486 15423 18523 10558 20075 20076 33453 33451 33450
33452 33456 33454 33455 32395 23029 31809 23027 23028 19519 19520 32550
19516 32551 19518 32549 14435 19517 23322 23317 22531 10537 21855 10535
23677 23329 23676 23315 6143 5437 5436 5927 5435 5438 4261 5113
5434 6302 6270 6332 5561 6418 4324 4328 4354 5153 6314 6490
4301 4783 6465 4472 4342 4859 10003 4218 4726 4863 4216 4230
4448 4741 4325 4337 4219 4573 4329 4411 4473 4330 4338 4213
4720 4513 4511 5389 5388 5165 4341 4351 4214 4514 5244 4232
5219 4451 4347 5218 5217 5216 4542 4231 4215 4343 4607 4340
5265 5148 4487 4217 4221 5146 5145 4452 5138 4353 5523 4483
4446 4574 5251 5252 5250 5247 5516 4786 4334 4333 4785 6464
6475 4705 5135 10005 4449 4947 6498 4925 4469 4413 4949 5997
4946 4944 4941 4939 4929 4930 4928 4194 4919 4918 4466 5144
32548 10534 23327 6476 4936 4934 4932 5478 4327 5143 4223 4943
4923 4484 23328 4727 4572 4150 5573 4942 4922 4339 4886 4455
4453 5248 4323 6392 4512 4445 5595 4945 4938 4415 4924 4352
4901 6093 23314 4900 33053 33203 4822 5553 6480 5481 4395 10536
4427 5537 4608 4447 10002 5524 5162 5482 5783 5156 5155 5139
5246 4920 4716 5140 4857 4719 4948 4940 4921 5480 5137 4926
5142 6462 5463 5141 5249 4937 5163 5479 4718 5452 4220 4412
6043 4419 10004 4931 4486 4474 4530 4478 4454 4485 4565 4935
5171 5416 5173 4731 4717 5136 4933 4326 5154 5164 5888 4605
4927 4737 6009 6451 5780 6198 5152 5477 4787 4784 4229 4450
4376 5476 5170 6466 6218 4703 31946 23324 23318 23320 23319 23312
23311 23310 23309 7552 8532 6616 6949 9402 8032 9026 8458 7430
7287 7549 8036 8034 7942 8162 8424 8699 8482 8879 8875 8872
8877 8873 8971 8874 8161 8845 8531 8605 9025 8876 8878 8917
8958 8033 7326 8970 7480 7663 9027 9322 7795 6918 8031 6999
7589 7789 7447 8208 7210 8349 8842 7415 8445 7414 8159 7417
8841 8511 7546 8867 8157 8928 8916 8580 9338 8843 6730 6710
8990 6594 15926 9585 585 32546 31354 23330 23321 21550 33630 713
16500 29798 16540 17866 17865 17714 17713 18410 18409 18408 18412 18411
18407 16614 16617 16615 16613 16616 16619 16618 31310 17936 20869 30926
786 32134 22556 32131 23107 32321 32322 23109 32133 32137 32323 32136
32135 23326
708: 33460 27410 13566 23325 23313 9952 23316 787 33627 814 759 31135
32308 17283 20352 22061 27408 27409 10257 26917 16131 25774 13883 15711
15699 14515 12870 17546 22487 23813 15779 23030 17000 16994 17788 17774
28088 16772 478 20503 11931 19229 21893 17135 18598 18072 16336 19099
12766 12790 14053 14052 19321 19851 19797 19551 24374 25881 30125 22322
33366 21666 24189 27175 25725 10661 13764 26496 26210 27207 9509 9505
16191 28394 28396 33180 28669 28668 17680 29309 22214 30013 30015 31392
14451 11816 13265 13264 31578 17603 10474 33620 10561 10560 10559 14277
10539 16625 9971 24627 19515 13000 13001 14795 871 907 906 905
1403 908 872 1825 1398 1402 1126 1545 1401 1153 1456 1251
1544 1376 1414 1527 1326 1382 1354 1494 1399 1370 1361 1341
1476 1587 1504 1817 1264 1623 1417 1639 1653 1543 1651 1590
862 1233 1715 1377 1400 33052 10540 23323 11598 23063 2641 3109
3386 3299 2319 3273 2961 2964 9770 2014 2635 2144 2963 2013
2015 2898 1853 10538 2993 2061 2105 9769 3272 2485 2214 2962
2718 2135 2142 2617 2139 2676 2490 2543 2257 2965 2289 2966
2277 3328 2134 2143 1928 2967 2310 2724 1939 2130 2491 1965
2140 2308 2128 2313 2193 1998 1850 2480 2131 1912 2192 1851
2592 2241 2212 4052 1849 2316 2245 1996 4048 2997 2968 2118
3086 2133 1839 2129 2297 2727 2486 2176 2479 4047 1894 2172
2076 2655 1947 4046 2208 4050 3066 2141 1892 1843 1913 2242
2179 1937 2251 2290 2749 2132 3023 4032 2373 4057 4069 1896
2492 2075 2279 2299 2171 2577 2320 2200 2155 2258 2180 4056
2174 1891 1966 4071 1865 4037 4033 1842 2000 3073 1863 4049
2191 1899 2779 1943 2712 2234 2383 9733 32132 23108 20083 18469
14945 10076 13455 19522 32011 32013 32012 16981 20107 20325 23293 13487
23019 14473 19901 13486 15423 18523 10558 10557 20075 20076 33453 33451
33452 33450 33456 33454 33455 32395 23029 31809 23027 23028 19519 19520
32550 19516 32551 19518 32549 14435 19517 23322 23317 10537 21855 10535
23677 23329 23676 23315 6270 6332 6418 6490 4301 5218 4329 5389
6465 4472 4449 4342 4859 10003 4218 4216 4230 4448 4741 4325
4219 4573 4473 4330 4213 4513 4341 4214 4514 5244 4232 4469
5219 4347 5216 4215 4343 5148 4487 4217 4221 5145 4452 5138
5523 4446 4574 5251 5250 5516 4786 4333 4785 6475 5135 10005
4726 6464 4334 5997 4946 4944 4941 4194 4919 4918 4324 4783
32548 23327 4328 6314 4354 6476 4939 5153 5144 5143 4337 4411
5388 5165 4351 4949 4943 4923 4451 5217 4542 4231 4607 4413
4340 5265 4353 4483 23328 4572 4150 6498 5573 4942 4925 4453
4863 4338 4720 4511 4947 4705 5146 4445 5252 5247 10534 5595
4945 4929 4930 4928 4327 4901 23314 4932 33053 33203 4822 5553
6480 5481 4466 4395 10536 5524 4936 4934 5537 5478 4886 5155
4608 10002 4323 4223 5482 5783 5156 4484 4727 4415 4924 4922
4339 4455 4900 5248 5246 6392 4512 4926 4716 5140 5139 6093
4948 4938 4352 5137 4719 4940 4427 4937 4447 6462 4857 4920
5452 5162 5142 5163 10004 4921 5480 5141 5249 5463 5173 4731
5136 6043 4933 4931 5154 4718 4717 4935 4927 5479 4220 4412
5164 4419 4454 4565 4486 4474 4530 4478 5152 4485 4787 4229
5477 4326 5888 4605 4737 5171 4784 6009 6451 5476 4450 5170
4703 4410 4704 4376 4222 6466 5558 6218 5310 6446 6463 6235
31946 23324 23318 23320 23319 23312 23311 23310 23309 8162 8699 8482
8879 8875 8872 8877 8873 8971 8874 8161 8605 8876 8878 8917
8958 8970 7480 7795 7447 8445 8159 8208 8841 7210 8349 8842
7415 7414 7417 8511 8867 8157 8928 8916 8843 6730 6710 6594
15926 9585 707 585 29277 32546 23330 23321 713 28700 29802 16529
29814 17866 17865 17714 17713 18410 18409 18408 18412 18407 18411 20529
31150 17936 32134 22556 32131 23107 32321 32322 23109 32133 32137 32323
32136 32135 23326 13167
709: 33238 13136 13961 14436 32531 32529 28326 23672 14437 23671 13964 23670
33354 25751 18535 28914 17254 28221 18115 20431 20423 23250 23252 23978
30596 30641 30609 31674 31673 31672 31671 1054 1196 1445 1967 3669
3230 29045 28801 29046 29097 31551 32401 28856 27128 14300 6151 4108
4780 7757 7624 7174 6659 8259 11223 33285 18505 18585 16546 16497
12156 12142 15882 11743 32464 33326 20114 15621 14318 11773 16623 24601
20067 33103 20133 16622 22965
710: 23116 23117 11331 25350 10634 23552 24538 26791 26667 22781 9795 23354
16727 28015 31921 28340 24121 10176 32983 17704 20086 19533 24132 19721
31561 20654 10970 10964 11263 16765
711: 1299 1644 3951 14574 1920 3321 2703 3479 3783 29346 28980 20280
11322 11321 19903 5254 5949 9409 8269 6881 10806 15442 20137 31117
712: 27438 32710 33070 9597 13551 28241 30221 11687 11686 32903 32926 33507
10497 14414 33571 18655 32103 32102 15215 13484 12594 17365 10899 12661
12701 31288 24857 13935 14211 20214 13006 10936 14538 23643 14358 14610
25308 32077 21093 14691 15138 14980 12915 12917 28230 15090 15494 15495
15552 14157 17495 22466 23819 15287 19927 15613 17688 16685 32368 15903
29870 11998 18441 18443 17183 17464 17244 18037 18045 18032 18738 16319
16381 24771 24707 18940 18938 18144 18216 19368 19809 19617 19991 25941
24405 9694 22975 20348 17979 22982 20937 25646 30752 30113 22724 22686
22670 22711 20801 20593 12474 12467 12480 24297 21664 23011 22851 22883
23253 23268 24051 25931 29972 27189 30184 26063 27477 25834 28833 27672
27771 25480 25463 10768 13741 13740 26336 26388 27928 27218 9476 9492
30619 30631 24582 17917 10840 10597 27018 17860 27996 17927 17928 28357
28316 27202 16950 14969 10327 17591 20220 23654 12482 26887 15744 27440
17074 23653 15611 14450 23720 29186 13845 17229 14109 23665 21559 13610
11659 24827 29033 22046 29129 28978 22045 32158 21850 31949 14288 11639
11436 10856 11132 21540 24828 25024 31394 31393 33304 26886 19280 28719
28335 20578 16553 12462 31165 31304 17902 21144 18658 30903 30936 30938
29212 24146 22203 17755 24939 9851 11145 9852 11037 11012 22832 32972
24933 32037 28047 12841 33624
713: 9564 31891 20532 13549 13544 12213 27613 27634 30264 30233 33557 31539
30097 10349 787 33621 570 567 33627 792 11157 13129 794 815
24995 17274 31505 759 32906 17681 11154 21671 17283 12537 19274 14517
18628 17993 27015 19997 32981 21108 22389 12757 32170 10340 30309 24134
14733 10248 16096 16112 27867 26917 11200 11202 14422 24094 31742 17406
12582 24838 24227 24243 25764 25167 11830 11452 11453 11454 21055 26552
13407 13413 19743 16918 18333 18339 12849 13942 13912 11720 13939 13228
14191 21027 18567 14366 14374 14369 14355 27120 15677 17725 23711 25201
25202 25200 14582 17071 32887 14670 14495 15195 15034 15050 12929 32292
32615 15114 25198 15564 14142 17507 22479 31779 15084 14458 32417 15773
23030 16992 19928 15523 17764 27902 28058 16294 29821 16291 28226 18794
31562 16778 16795 26001 16857 32374 667 478 16823 16882 20494 11979
19205 21877 17182 17108 18599 12732 18080 28584 18847 11158 18711 18682
16363 16337 24773 24696 18972 19089 28909 33618 33615 12066 9895 18163
14046 14023 19336 19878 19798 23490 19508 19621 19760 20466 20464 20017
21927 15746 24996 24362 9692 28859 20976 20983 14104 20562 26653 25607
25544 30116 30127 30112 22779 21720 20783 32191 23181 12342 30320 22317
12315 33375 21687 22951 22219 22842 21472 21384 21220 24029 25914 24065
28261 27200 27176 24002 25729 25735 26059 24653 25867 27475 25095 26601
24291 28272 25131 26958 26964 25672 27696 27765 25323 13031 10735 10742
10659 10685 10744 24960 27102 27110 27055 25261 31701 31675 31518 31052
26492 26363 26278 27945 27935 27974 27242 27250 11380 11375 9515 9473
9513 12033 11463 29780 30630 12384 13328 13334 26095 16217 12533 10372
30513 30505 30510 30515 26027 16070 16042 16054 12613 12229 14942 14938
10830 28020 27984 27333 18003 28375 28397 28140 28471 28477 28309 28313
33180 28669 28668 17680 29309 30767 32130 17432 32128 10939 33561 30013
460 13663 21930 17626 20531 12488 18504 24025 18461 11816 14447 16397
13265 13264 31578 17603 10474 33620 10539 17580 32280 28644 15941 1416
1019 33052 32038 17665 10540 27443 477 23063 10628 1870 2733 1871
2731 3016 2325 2732 9733 20083 18393 18469 14945 19522 22158 32011
32013 32012 29143 20325 23293 33029 14473 14567 14566 9789 15423 33453
33451 33450 33452 33456 33454 33455 32395 23029 23027 23028 11306 19519
19520 32550 19516 32551 19518 32549 14435 19517 10463 5117 4724 6281
5774 6282 5220 9079 8640 8194 20355 15926 22107 22108 9811 11814
23747 707 585 31913 20720 23708 32546 10207 30746 31907 31906 31908
33276 9965 14409 21551 23827 23828 28706 23742 16577 29810 16520 29803
16554 29812 12460 31198 31172 20526 32050 27143 31313 31329 31299 20882
20879 20877 19169 21523 30895 22109 12096 12949 10617 21766 21515 16591
12168 708 26874 31407 31438 13164 13191 13193
714: 33138 17600 31235 12431 30249 30285 30286 30229 13110 19705 20735 21009
28880 28233 14320 32870 28877 28878 28879 31859 22093 18513 18512 15641
22819 33474 32168 9847 33174 13257 15229 10066 11738 17379 30002 13390
15135 14975 15563 9761 16892 20510 11944 29595 18675 16344 24710 18936
18116 18219 19815 23444 23545 19652 24425 33347 21543 21544 30110 32203
23214 12280 12316 12313 22206 22285 22839 21451 21247 23157 27181 23943
30183 25102 30028 26945 25226 25474 27071 27041 13744 26514 26286 27963
11511 29755 16475 13312 16186 26155 26146 24587 31008 31010 28360 32879
21105 9956 18629 16433 29280 6404 5020 4401 4357 7870 16952 21803
15606 16555 9841 30956 25383 820
715: 30770 29833 14238 22930 20888 1431 1487 1552 1026 1557 1488 4074
3289 3288 4073 2021 1860 2338 2541 33067 21985 29078 22050 31542
16917 13448 16634 30864 4180 4181 6190 6140 6343 5755 4386 4852
5102 4850 6344 4851 4433 31899 7978 8185 8187 8718 7561 9140
8717 7297 8950 8951 8497 8186 9006 6852 8801 6588 8561
716: 33046 22935 23438 24485 26803 25612 22760 31786 11130 9443 10427 22821
27528 10946 32385 1066 1815 1209 11284 25385 2427 3995 3996 20060
32139 17435 29230 17076 10226 24793 15295 9926 13231 33172 20691 9924
22954 27461 29154 29157 20611 13475 15234
717: 1182 2509 3587 5318 5643 8265 8871 6566
718: 10217 11761 881 3489 3484 3523 29398 32014 5496 5297 5404 4584
4437 7344
719: 30392 24618 1062 28936 5945 6411 4286 6995 2439
720: 13578 23325 23313 9952 23316 17221 13111 759 31135 17283 22061 26917
25142 15808 23030 28211 33625 25879 31058 33180 28669 28668 17680 29309
30013 30015 31392 14451 11816 13265 13264 31578 17603 10474 33620 10561
10560 10559 14277 10539 16625 9971 24627 19515 13000 13001 14795 871
907 906 905 1403 908 872 1825 1398 1402 1126 1545 1401
1153 1456 1251 1544 1376 1414 1527 1326 1382 1354 1494 1399
1370 1361 1341 1476 1587 1504 1817 1623 1264 1639 1417 1653
1543 1651 1590 862 1233 1377 1715 1400 33052 10540 23323 477
23063 2641 3109 3386 3273 2964 9770 2014 2319 2635 3299 2144
2961 2963 2013 2015 2898 10538 2993 1853 2061 2105 9769 3272
2485 2214 2718 2135 2962 2142 2617 2139 2676 2490 2257 2965
2543 2966 2289 2277 3328 2134 2143 1928 2724 1939 2130 2491
2967 1965 2140 2308 2313 2310 2128 2193 1998 1850 2480 2131
1912 2192 1851 2592 2241 2212 4052 1849 2316 2245 1996 4048
2997 2968 2118 3086 2133 1839 2129 2297 2486 2176 4047 1894
2479 2172 2076 2727 1947 4046 2208 4050 3066 2141 1892 1843
1913 2242 2179 1937 2655 2251 2290 2749 2132 3023 4032 2373
4069 1896 2492 2075 2279 2299 2171 2200 2577 2320 2155 2258
2180 4056 2174 1891 4057 1966 4071 1865 4037 4033 1842 2000
3073 1863 4049 2191 1899 2779 1943 2712 2234 2383 9733 32132
23108 20083 18469 14945 10076 13455 19522 32011 32013 32012 16981 20107
20325 23293 13487 23019 14473 19901 13486 15423 18523 10558 20075 20076
33453 33451 33450 33452 33456 33454 33455 32395 23029 31809 23027 23028
19519 19520 32550 19516 32551 19518 32549 14435 19517 23322 23317 10537
21855 10535 23677 23329 23676 23315 6270 6332 6418 4324 4328 4354
5153 6314 6490 4301 4783 6465 4472 4342 4859 10003 4218 4726
4863 4216 4230 4448 4741 4325 4337 4219 4573 4329 4411 4473
4330 4338 4213 4720 4513 4511 5389 5388 5165 4341 4351 4214
4514 5244 4232 5219 4451 4347 5218 5217 5216 4542 4231 4215
4343 4607 4340 5265 5148 4487 4217 4221 5146 5145 4452 5138
4353 5523 4483 4446 4574 5251 5252 5250 5247 5516 4786 4334
4333 4785 6464 6475 4705 5135 10005 4449 4947 6498 4925 4469
4413 4949 5997 4946 4944 4941 4939 4934 4929 4930 4928 4194
4919 4918 4466 5144 32548 10534 23327 6476 4936 4932 5478 4327
5143 4223 4943 4923 4484 23328 4727 4572 4150 5573 4942 4922
4339 4886 4455 4453 5248 4323 6392 4512 4445 5595 4945 4938
4415 4924 4352 4901 6093 23314 33053 33203 4822 5553 6480 5481
4395 10536 4427 5537 4608 4447 10002 5524 5482 5783 5156 5162
5155 5139 4900 5246 4920 4716 5140 4857 4719 4948 4940 4921
5480 5137 4926 5142 6462 4220 5463 5141 5249 4937 5163 5479
4718 5452 4412 6043 4419 10004 4931 4486 4474 4530 4478 4454
4485 4565 4935 5173 4731 4717 5136 4933 4326 5154 5164 5888
4605 4927 4737 5152 5171 6009 6451 5477 4787 4784 4229 4450
4376 5476 4410 5170 6466 6218 4703 5558 6463 6235 5310 4704
4222 31946 23324 23318 23320 23319 23312 23311 23310 23309 8162 8699
8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 8876 8878
8917 8958 8970 7480 7795 7447 8208 7210 8349 8842 7415 8445
7414 8159 7417 8841 8511 8867 8157 8928 8916 8843 6730 6710
6594 15926 9585 707 585 32546 23330 23321 16504 17866 17865 17714
17713 18410 18409 18408 18412 18411 18407 17936 32134 22556 32131 23107
32321 32322 23109 32133 32137 32323 32136 32135 21513 21514 23326 33622
721: 32248 32245 24862 11228 33098 32473 13108 13143 27853 23349 23678 10290
10319 10312 16102 29573 11206 17367 12572 9664 25750 21086 13374 18343
11707 13850 13898 14194 18561 14370 14376 15714 14598 14591 15164 15009
15128 14143 14120 22441 23769 14456 15797 30982 30979 17833 27909 28096
28084 16678 16661 16851 27580 16835 20512 20513 11947 11949 19254 19257
19250 19265 21114 21113 18596 28518 28615 16366 16380 18935 12776 12803
9879 18158 14014 13797 19323 19808 23715 19644 19686 19660 19634 20472
20474 20463 25058 20815 32192 12279 33422 22225 27178 27187 23920 25168
26171 26062 25100 30055 28275 25130 26934 25695 25208 25709 27753 25427
10769 10778 10771 27098 27036 13765 13768 31069 26508 26331 26201 27925
27959 9489 9501 9530 32667 30604 13300 10407 30514 16072 12612 10975
12264 28022 28366 28481 28315 10928 16038 15088 16436 16450 32835 14788
15726 14787 33274 15429 13521 32652 16979 16725 31370 32553 1741 1596
1308 1070 11573 11615 14950 27604 1908 3553 2715 3010 2867 3600
4012 3880 3059 33611 4013 2256 2717 2326 2387 2224 2226 3118
2728 3021 4011 2881 3494 2589 4014 2668 29241 22009 29111 31471
22967 14408 14954 16903 16977 19712 4561 6519 6520 4312 5383 4196
5810 5832 4974 4649 5380 5381 6293 5972 4973 4972 5121 5382
4428 4375 4569 8469 6665 6656 7872 7189 8515 8984 8356 7873
8357 8355 8239 7321 6660 7998 8663 7874 6893 7118 8514 8359
8983 7721 6555 8358 8064 6843 8609 7738 9231 7423 7220 8450
7047 25232 15582 15575 9781 13515 28790 31384 14827 20579 24903 9724
23039 33306 31576 17611 20892 16528 31213 31154 20878 28249 10616 21482
31414 30997 13202 30996 23085 13209 13196
722: 28805 23584 24344 26726 25623 22755 1699 1327 1681 1711 30758 3368
2948 3888 3886 3887 2978 32751 9598 4464 5090 5078 6170 6064
6545 6540 4715 5077 5091 4479 9156 6990 7313 7304 8320 8319
7778 6951 6716 23002 20616 24810 17878 32560
723: 31932 31934 19461 20081 20080 19723 14967 14630 14957 14274 14275 20923
14629 20924 14276 31933 3398 3490 28994 29020 20306 14571 6922 8006
7966 7965 9049 28803 31935 15387 15729
724: 14296 22529 33340 30796 30810 20033 20027 20031 30802 31638 9847 30791
30811 20028 30823 20025 20029 28808 1714 30783 30789 30792 30793 30784
30803 30815 30816 30818 30817 30804 33111 3859 29100 13480 13481 21988
22023 30788 30787 30775 30779 30785 30780 30777 30778 30776 30795 30800
30798 30799 30797 30812 30813 15073 30814 6124 30822 7786 9318 9370
6609 9317 30805 30820 30786 30781 30819 30808 30807 30809 30806 10067
14821 11217 30801 30794 30821 30782 30790 20030 20032
725: 1478 2073 9187 7656 7243 9555 33165 15862
726: 11558 11559 31546 28634 29412 14396 979 24942 26615 21773 11102 11098
11106 11107 11105 11108 11103 11099 11097 11096 11101 11100 11275 30394
11551 27445 27436 21094 24600 22619 22620 33296
727: 13632 33290 15759 33058 28237 28239 28238 29617 29611 29623 29620 29619
29618 29616 29615 29614 29612 29610 29605 29601 29613 29607 29602 29622
29621 29603 29608 29606 29604 29609 26912 15585 26908 26909 14819 24142
16629 23764 24905 15293 19916 25354 25353 15271 13616 32040 32041 32039
22500 32621 16040 16302 33309 29885 11888 18894 20002 23453 9973 29238
20331 21808 23297 10192 14841 14840 14842 20852 18653 33312 21738 28659
9945 20156 14112 11149 20442 21136 11059 23119 13787 23408 23409 13284
20733 14762 17627 33518 15958 22080 31614 27170 14330 10942 30547 29214
30486 33069 22806 30534 13614 21740 9981 22120 20024 22553 20023 22121
22929 17974 16028 18654 987 28881 19424 17662 18765 29278 24852 24945
32675 33249 30458 30457 30459 30460 25381 19145 2931 3359 2913 10439
13628 14188 20056 26900 14441 22505 33051 22907 32138 29634 11795 28862
28861 31661 31660 28863 20447 16928 14410 14411 31382 31618 18643 24920
31767 13456 23285 31815 11313 11314 24929 24928 17703 20732 11308 20958
20957 25506 11316 11317 20731 21578 21579 30863 20967 32978 10920 10921
10922 10923 22201 19472 19471 11456 20265 24037 13784 32888 32889 12746
26825 19191 28640 19190 16935 23120 30545 28855 20438 32243 27446 23719
11142 18460 20637 14448 18435 11442 22497 28802 29169 19435 29675 10335
32044 24038 31929 9742 9743 9741 33291 20730 31883 24602 33280
728: 18117 19479 30364 11771 22938 1201 3757 2877 3313 31466 21993 30834
6322 5462 5641 5461 4170 4253 5648 5983 30845
729: 13735 27875 16141 25156 12652 12693 17554 19772 21693 16241 17158 16361
23544 26631 26728 30134 20802 25927 31087 14886 29266 20144 18652 1637
1032 1356 1433 28954 31924 11141 6403 5494 9982 20249 30719 24949
14438 30683 9791 12186
730: 14764 23303 22414 1242 1146 1854 3516 22493 30200 29054 26906 10877
15331 14472 14471 22912 5789 8190 7532 27445 27436 18524 18862 29323
731: 22988 1463 1536 1468 3764 2442 3715 3411 2190 2441 3243 2830
9582 9581 29317 28898 30467 5664 6173 4172 4861 6244 4697 6187
4128 4101 5831 4545 4414 8554 7629 7329 6804 8477 8206 8189
9077 6797 9019 8039 7842 7970 7781 7782 6610 7780 6704 6898
732: 10502 33256 25807 13112 10050 33595 10460 10261 30296 29570 29425 17385
17315 12581 11114 13398 13406 12677 12682 12713 12719 18322 13853 12859
18566 14525 17723 14478 15139 14981 32296 19012 15549 14132 14161 22474
30962 17035 17781 16285 16715 27555 27563 16808 16891 15915 11982 11969
19251 19221 17480 17109 18611 18085 18083 28613 18747 18746 18680 16376
24831 24774 18959 19083 19086 9896 17964 18223 13978 19387 19403 19879
19811 19691 25951 24422 24406 33599 20989 26717 25527 20793 32222 22335
22380 12309 21638 21592 22218 21331 24187 23148 28255 27180 30185 30172
25792 24663 30037 25671 27736 13028 10759 10694 30157 24971 27118 27062
31030 26400 26414 26202 26535 11326 9479 11489 11481 16495 13320 13327
26157 10398 26040 14931 28202 28195 28419 28147 28460 11093 20947 15369
9433 22532 19727 18659 16421 16424 28002 21116 21115 31641 21174 1613
1811 9786 27531 3508 4029 18510 27203 24904 22829 17660 20309 29116
29115 13727 10657 13960 33552 10173 10172 10174 31005 4289 5617 8286
9215 8732 6723 9216 15973 31356 31357 28918 20682 10541 15360 29865
30826 30825 16976 15056 15057 15055 20003 9962 29639 14314 16499 31214
9821 20843 30550 18993 30445 27000 31613 31435 13177
733: 3978 2546 2707 3977 19195 29017 4346 4345 6567 7255 7155 9308
7512 6840 7269 9310 9309 8575 9106 7585 6783 6599 17276 17622
26579 26578
734: 17632 13559 13562 27605 32326 25068 13120 13133 13106 24941 10284 10283
29185 16093 30291 29580 29953 17321 17337 12549 24242 24241 25139 25135
25166 21085 13902 18547 15956 14357 15681 14618 14602 17052 15145 15040
12942 26163 15097 15125 15529 15565 14127 14153 22446 15764 17008 17805
17804 28094 28216 28225 18784 16773 32354 32352 29852 29880 11968 12017
11945 19217 19218 24105 22086 18588 28542 18898 18948 12063 9892 18206
14051 14005 19322 19297 19482 20021 20558 25889 25033 25044 21729 20777
12469 23193 23215 30335 12302 33363 21642 22256 23269 23154 24071 30535
19285 24283 25788 25704 27745 25444 10746 25290 31031 31048 26504 26426
26473 26516 26491 26227 26346 26349 27941 27936 11523 11524 11522 30633
26099 16183 16184 16172 30523 26019 16080 12609 12608 18637 27364 27343
17650 14637 20842 22077 22205 11857 11642 20247 31125 11135 10001 25502
29691 13268 14885 11125 16975 9040 10209 30754 30487 27310 27309 10034
15086 20276 9920 12590 20277 10032 10033 20278 23655 27525 22548 14240
24021 10031 11411 30400 31874 20279 10025 10026 10024 10041 10040 10035
10030 10027 10028 10038 10037 10029 10039 10036 13249 31111 13247 23077
28678 29816 29797 16539 13235 9673 15505 14839 9816 15877 31311 20870
33332 33066 21521 30894 31598 29979 12971 21486 16584 12161 26842 18430
28052 23083 13179
735: 27027 31939 25235 25234 25249 25240 27839 23239 14560 14559 14095 14558
14557 14096 14724 30565 30585 30571 30568 30576 30578 30577 30570 30569
30586 30563 30562 30573 30572 30579 30574 16097 16123 11192 16135 10885
12548 20091 12270 14814 14813 14812 14815 14810 14236 13427 12681 12718
18318 14331 18546 14523 16003 16004 16005 16006 16008 16007 14483 15169
15013 32628 19003 16261 25248 25247 16667 16666 16878 13636 21884 23695
17164 17456 18615 18724 16371 31958 12825 25237 25236 25239 25238 25241
25242 25250 25251 19393 19318 19882 20552 21721 12361 10085 12308 30566
23378 24198 30174 26085 23168 28832 30146 24979 27064 25300 27161 27248
9523 11510 12415 13346 26158 26149 17916 14923 28204 18004 28456 25246
25245 27022 27024 27025 27023 27026 27028 27029 25243 25244 29678 917
1076 1586 1109 915 10478 3708 3455 4058 3050 2451 2930 3703
3373 3911 3297 2401 2057 3674 3158 3401 1993 2575 1901 1994
1995 2380 10873 14816 14817 19358 17285 32471 5401 5501 6031 5402
6032 4502 5442 4235 5110 5455 5502 5403 5457 5903 5705 4400
5883 5589 4315 5266 6432 4844 5993 7598 8241 7571 8242 6981
7139 8705 8945 9234 8094 7948 6980 6791 7766 18426 22406 22405
22409 22410 10520 22397 22396 22407 10518 10519 33441 33438 33445 33443
33442 33440 33444 33437 33439 31488 29411 29410 22398 22400 22399 22411
22401 22408 22402 10521 22403 22404 27020 27021 19908 10523 819 17679
16923 17678 16921 30575 30582 30580 30584 30583 30581 30564 30567 16919
16920 16922 31202 10436 10435 10433 10432 10431 10434 20444 20445 20443
20446 29166 29167 29164 29163 29165 23389 23384 23383 23373 23372 20824
20093 20825 20096 10468 29740 20098 20826 20101 20095 19921 10470 29739
15663 10472 19920 20100 10471 10469 20099 20094 29414 20097 23375 23377
23376 23374 23385 23386 23380 23379 23381 23382 23387 31612 23388 10522
23390 31610 31611 19910 19909 22412 21759 29155 26876
736: 1113
737: 22165 24085 17340 21917 15184 15025 15107 17518 22490 22450 23789 15279
16852 27585 11935 17148 17128 18070 18057 28612 24744 18922 19042 19047
18138 20477 20465 20968 32196 12345 30351 22379 33413 33400 21617 21421
21460 21308 21326 21242 25922 25929 27193 23909 26069 24660 30070 25829
27668 27709 27809 27801 24978 27211 12494 10387 25500 27347 28371 28122
31012 13683 31864 29699 1141 1275 3296 3932 2018 2996 2637 22032
29318 28985 4379 5959 5844 5076 5843 5842 9053 7523 7428 8745
8563 28715 21095 23674 31218 22164 30913 12159 15393
738: 33464 10059 18656 16009 31629 19719 15168 15012 23770 23761 27922 28083
32355 19198 33211 18752 18754 18755 18900 18111 18194 23564 23505 23491
23591 23455 23488 23636 23470 23614 23496 23495 23620 19587 24513 24337
24375 24334 24528 24454 9684 9686 9693 9710 20426 26774 26813 26773
26643 26704 26786 26652 26639 22705 22758 22681 22767 22700 22738 22652
22654 20804 23984 23994 25664 26378 11492 11491 30671 12397 16163 12267
12240 11251 10850 11245 10825 28188 28192 28012 19765 29729 29735 29680
19469 19767 19768 1561 1523 1458 1292 1135 999 1137 1460 1609
19710 26915 19709 29281 11607 3116 4004 3115 26884 22057 11651 31366
31367 20205 14633 4891 7162 9196 9406 9820 32249 9819 9919 31703
20664 20651 13152 20649 28812 33551 18000 33278 28752 16506 29162 29161
29160 29159 29158 18296 11042 12149 13215 13187
739: 13469 11120 1529 1640 1530 3771 2705 3155 3570 3179 2514 29025
6305 4133 6390 6391 4614 5289 8931 8494 7750 8737 8476 9311
7767 7728 9312 8366 8738 7621 7620 8913 8865 13637
740: 1283 1548 2548 3641 3018 2545 5974 4997 6051 5001 9091 8631
9364 8632 9362 8633 7515 6903 7296 6595 33481 15573 33164
741: 12202 27619 31857 17431 15605 16105 16114 24087 12545 21087 12660 14200
18543 14346 15675 17073 15207 15046 16991 15513 17762 20505 11933 19203
18614 12738 28586 16339 18916 12839 12049 9910 19888 23605 24393 9687
26692 26810 22731 23220 12338 30333 22314 12290 29215 25716 27485 30073
24293 28287 27700 27674 27769 24984 13769 25265 31054 26451 26344 26293
27304 9532 11509 16492 30506 14912 10824 28414 28229 22962 20108 30375
30373 22960 29685 1708 1635 16911 16910 16912 32084 32085 31119 31120
31121 2103 3964 2615 3924 3965 23723 23724 29334 21973 12193 12194
22083 31856 31855 30374 23887 31858 24117 24119 24118 21131 32283 32282
28906 32281 32284 18466 11701 22813 31118 4985 4987 5642 4986 6749
8008 9275 8576 7459 8461 33517 10211 10872 11060 11061 24791 19152
14166 31225 31290 20860 21525 31997 21518 31381 18626 32427 30696
742: 10652 26605 10189 29336 26608 32646 32645 24953 26604 31901 15972 14237
21168 21170 21169 22097 27430 33573 33572 32943 23034 17894 12428 28341
15348 15347 31646 17592 33594 31649 31648 32935 31922 20728 23033 10203
23032 1036 10857 17252 11170 3661 3651 3806 2584 2278 32640 32641
32642 32636 32637 32638 10064 32644 32643 32639 22550 28916 28927 29035
29245 28896 28938 28945 28895 21942 28946 29260 29261 29347 29244 22054
29060 29027 28957 20285 20838 21098 23031 26524 31654 18630 32536 18402
31645 31644 23663 10224 18437 31551 13974 17977 6091 6292 5190 5046
7868 6693 7156 9247 7511 6718 6612 9384 7402 7599 8512 6574
7540 8820 8540 13782 13781 23398 13286 26992 11223 16652 31911 14189
22105 13447 9969 22117 15981 25196 10932 10931 10930 10933 10934 26897
24621 11009 14832 26899 26895 26898 26896 15863 11778 24611 32840 16986
18859 24620 18860 15859 15733 11777 31980
743: 17215 23122 30256 30255 25808 13090 30853 30859 30861 30850 30852 30851
30858 30860 16000 21796 10109 10104 10105 10106 10111 10117 26559 21787
23123 26564 15989 24888 24890 24889 25762 21082 33024 14995 12866 15566
23803 15282 10103 26569 26568 28059 26555 21789 24848 32309 32159 28601
19331 19583 24496 26563 15987 25578 30117 20773 30359 21797 21733 21731
21732 26560 21286 30054 30078 28847 31082 12036 12408 16181 13271 18638
14935 32965 32232 18002 18008 21786 21785 15994 21783 16001 15750 31625
15660 10193 24599 15658 15659 17900 31620 1718 981 16089 2185 3780
2776 3781 2947 3713 1930 2238 3167 3692 1862 10461 29081 14432
26554 30856 30854 30855 30857 4321 4282 4507 4475 4613 8895 7717
7516 8634 8415 8894 9000 21794 21799 20653 26556 21798 24870 24869
24868 24871 24872 10135 10138 10136 10143 10140 10139 10134 10144 10141
10132 10145 10142 10137 10133 24873 24112 24111 24113 24895 24894 24874
24876 24878 24875 22116 17671 24877 17691 17690 17674 17672 17673 17670
17675 24879 24880 24882 24881 24883 24887 24885 24886 24884 21734 21735
24893 24892 24891 24897 24896 24899 24898 24901 24902 24900 16544 21792
15990 21784 10082 26561 10094 15986 15992 15991 26567 15988 26565 15984
26566 15983 26562 21801 26570 15993 10122 10107 10121 10110 10123 10108
10120 26558 10112 10113 10119 10118 21790 10115 10114 10116 21503 10129
21793 26553 21800 10128 21795 21788 15998 15999 26557 21791 31601 31621
26838 16940 16941 22557 16984 31369
744: 23613 23472 24403 24451 26790 26776 25659 25549 22754 22667 1808 1272
1670 1802 1803 3960 2893 2475 2199 2496 2384 3584 3949 3078
3277 3947 3948 21965 20315 19359 15217 24938 6373 5655 5666 5583
4186 6052 4730 6458 8578 6738 9918 9315 8211 9162 7137 9316
9917 7995 8090 7547 8756 8091 7138 8212 8729 7912 7994 9033
8214 8213 7136 7113 32316 22923 20718 14768 14767 19915 20191 14797
745: 24910 1785 1794 1611 1372 1271 900 1612 1751 1610 1638 17231
33031 7142
746: 25627 25545 21171 14739 1000 3720 2977 3182 3547 3506 4076 2167
17676 31109 28899 28900 29201 26176 15574 6260 5957 5799 4905 7058
8408 8144 7291 9103 7996 7064 32110 29172 32253 16915 16914 14807
747: 30237 10189 29336 26604 15972 28209 28594 23489 23626 24440 24410 20405
26799 25034 22630 23250 23252 23934 25439 10736 13227 27353 13971 30539
30538 31338 990 924 961 1573 1175 1405 1194 1856 13631 14064
32639 15931 15930 21999 21998 26916 14065 14063 31645 23663 30841 18437
13974 6426 5054 8684 6615 6957 8722 7820 7046 7818 7819 6638
7779 26993 16652 20625 9588 20712 15925 15581 23023 23024 28501 19465
17933 19726 19466 17938 32562 19464 31257 31975 32858 32863 32945 31013
24840 10216 627 11742 9553 17883
748: 33256 29367 10050 33595 10460 17385 12677 12713 17723 15549 17109 19083
18223 13978 19387 19351 25951 24422 24406 33599 26717 25527 22636 22692
23148 28344 30185 26595 10759 30157 16495 9433 30019 22532 20000 33305
18659 28002 27013 31641 24946 11265 21174 1057 32996 22983 25336 15462
2654 2026 2052 3077 3878 3235 17233 33463 10865 29226 24993 22829
22828 17660 13727 10070 13960 10172 10174 10173 33356 31005 9028 11875
22213 25360 20682 18397 18396 32254 30826 16976 10245 14314 32049 20843
20941 20944 20945 20943 20946 20942 31799 20349 30445 27000 14809 14747
749: 32565 33633 25403 25404 20151 18429 30313 18448 27873 11190 17414 24232
13429 18313 13907 32607 28853 28069 16897 19263 22514 18592 16373 24776
19108 12830 19332 19885 20341 20566 22675 10091 33364 21645 23130 29966
23170 28280 26980 27112 27155 26189 27954 27961 9525 11476 18650 29760
16489 13348 17909 13273 16082 10591 19107 22910 28451 16409 29684 887
1199 1198 1350 1374 11451 3260 3894 2146 2462 3261 3893 2904
2581 5352 5580 5343 5348 5349 5350 5581 5347 5304 4292 5042
6026 6250 5977 5712 5293 20605 5453 5902 5292 5341 5305 5345
5233 5210 5338 5344 5340 6486 7351 9319 7641 7643 8257 8700
9009 8862 7642 7644 9320 6787 8701 6565 8085 7799 7029 8084
7331 6569 8258 8863 12453 31206 20585 27010 31605 21770 16598 12179
26854 31400 28050
750: 10320 10264 25900 17334 14217 17034 17003 17001 17846 17787 27579 29845
29846 16880 29868 19255 17081 18645 28541 24753 12070 23576 19578 25954
20545 23184 30358 21659 23959 23914 26173 24644 24675 25859 30033 25215
27806 27754 13019 26493 26453 26231 26309 27967 29776 29777 29768 30612
30678 26139 10371 28124 28148 10073 20069 29702 16413 6816 8965 17898
18798 17596 15459 17595 14758 17569 28913 32960 9960 21117 22186 12993
12962 10623 22994 28854 13244 26839 26865 26872 17594 13201 13168 17593
751: 33072
752: 31554 30287 30288 30284 30259 9970 9825 16002 27864 10910 12644 12688
14550 19036 14145 22417 28082 15908 21860 17471 18036 19138 19652 22298
22839 21358 21360 21359 9529 29749 16165 16170 16180 26148 12241 28135
28136 11444 1009 1048 3360 2795 3718 2012 2816 2366 2815 2817
2365 2342 2067 2322 2263 1888 28334 20953 20954 20951 20950 20955
20952 15264 15265 5073 5072 6503 5723 4461 28333 6502 6181 5308
4138 5309 8121 7499 9211 9376 9141 6742 7349 7222 7891 8243
7698 9392 7699 7923 7924 8506 20274 20273 20271 20272 7610 26527
26526 11113
753: 1179 1491 1877 22044 5528 4123 5467 5468 9238 8163
754: 22815 10867 29459 20918 21112 1688 1378 29184 10511 2531 22501 4252
5545 4121 4120 5631 6541 4910 7683 7969
755: 21160 11331 23462 24537 24408 26791 26667 26756 25617 25636 22766 22643
32043 930 2489 2370 3794 3190 2369 2643 3076 3793 2307 19273
29003 19902 5758 6197 7060 9059 7375 8486 7295 8487 8847 32108
30498 28893 32251 31775 20711 32583 31331 14837 33476
756: 13533 23325 23313 9952 23316 13126 31135 17283 22061 26917 25753 25145
21058 21078 21053 21060 15792 15812 23030 667 478 33625 30120 25032
22221 22223 22268 31084 31077 33180 28669 28668 17680 29309 30013 30015
31392 14451 11816 13265 13264 31578 17603 10474 33620 10561 10560 10559
14277 10539 16625 9971 24627 19515 13000 13001 14795 871 907 906
905 1403 908 872 1825 1398 1402 1126 1545 1401 1153 1456
1251 1544 1376 1414 1527 1326 1382 1354 1494 1399 1370 1361
1341 1476 1587 1504 1817 1623 1264 1639 1417 1653 1543 1651
1590 862 1233 1377 1715 1400 33052 10540 23323 477 23063 2641
3109 3386 3273 2964 9770 2014 2319 2635 3299 2144 2961 2963
2013 2015 2898 10538 2993 1853 2061 2105 9769 3272 2485 2214
2718 2135 2962 2142 2617 2139 2676 2490 2257 2965 2543 2966
2289 2277 3328 2134 2143 1928 2724 1939 2130 2491 2967 1965
2140 2308 2313 2310 2128 2193 1998 1850 2480 2131 1912 2192
1851 2592 2241 2212 4052 1849 2316 2245 1996 4048 2997 2968
2118 3086 2133 1839 2129 2297 2486 2176 4047 1894 2479 2172
2076 2727 1947 4046 2208 4050 3066 2141 1892 1843 1913 2242
2179 1937 2655 2251 2290 2749 2132 3023 4032 2373 4069 1896
2492 2075 2279 2299 2171 2577 2320 2200 2155 2258 2180 4056
2174 1891 4057 1966 4071 1865 4037 4033 1842 2000 3073 1863
2712 4049 2191 1899 2779 1943 2234 2383 2680 1841 2164 9733
32132 23108 20083 18469 33178 14945 10076 13455 19522 32011 32013 32012
16981 20107 20325 23293 13487 23019 14473 19901 13486 15423 18523 10558
10557 20075 20076 33453 33451 33450 33452 33456 33454 33455 32395 23029
31809 23027 23028 19519 19520 32550 19516 32551 19518 32549 14435 19517
23322 23317 10537 21855 10535 23677 23329 23676 23315 6270 6332 6418
4324 4328 4354 5153 6314 6490 4301 4783 6465 4472 4342 4859
10003 4218 4726 4863 4216 4230 4448 4741 4325 4337 4219 4573
4329 4411 4473 4330 4338 4213 4720 4513 4511 5389 5388 5165
4341 4351 4214 4514 5244 4232 5219 4451 4347 5218 5217 5216
4542 4231 4215 4343 4607 4340 5265 5148 4487 4217 4221 5146
5145 4452 5138 4353 5523 4483 4446 4574 5251 5252 5250 5247
5516 4786 4334 4333 4785 6464 6475 4705 5135 10005 4449 4947
6498 4925 4469 4413 4949 5997 4946 4944 4941 4939 4929 4930
4928 4194 4919 4918 4466 5144 32548 10534 23327 6476 4936 4934
4932 5478 4327 5143 4223 4943 4923 4484 23328 4727 4572 4150
5573 4942 4922 4339 4886 4455 4453 5248 4323 6392 5162 4512
4445 5595 4945 4938 4415 4924 4352 4901 6093 23314 33053 33203
4822 5553 6480 4419 5481 4395 10536 4427 5537 4608 4447 10002
5524 5482 5783 5156 5155 5139 4900 5246 4920 4716 5140 4857
4719 4948 4940 4921 5480 5137 4926 5142 6462 5463 5141 5249
4937 5163 5479 4718 5452 4220 4412 6043 10004 4931 4486 4474
4530 4478 4454 4485 4565 4935 5173 4731 4717 5136 4933 4326
5154 5164 5888 4605 4927 4737 5171 6009 6451 5152 5477 4787
4784 4229 4450 4376 5476 5170 6466 6218 4703 4410 5558 6463
6235 5310 4704 4222 6446 6400 6003 31946 23324 23318 23320 23319
23312 23311 23310 23309 8162 8699 8482 8879 8875 8872 8877 8873
8971 8874 8161 8605 8876 8878 8917 8958 8970 7480 7795 7447
8208 7210 8349 8842 7415 8445 7414 8159 7417 8841 8511 8867
8157 8928 8916 8843 6730 6710 6594 15926 9585 707 585 32546
23330 23321 33630 713 16572 16501 17866 17865 17714 17713 18410 18409
18408 18412 18411 18407 17936 32134 22556 32131 23107 32321 32322 23109
32133 32137 32323 32136 32135 21492 23326
757: 914 1005 1155 1677 1664 893 1351 1662 1798 3639 3640 3574
3593 3189 2346 6474 4654 5448 9142 6551 6866 7184 8519 33612
758: 1483 3457 3452 2938 3138 2924 2515 30398 21976 29094 5567 6405
5074 9073 8341 15399 15390 25252
759: 32258 27308 14845 32318 30725 13571 13565 14734 17631 14114 10426 12201
27633 11054 17932 30258 30242 15884 25804 15647 25801 824 33627 567
756 33628 586 787 570 720 33621 792 628 613 815 13094
10546 814 33619 794 32919 32918 10600 11112 18499 18500 25401 17283
31634 27824 27849 27823 22611 12541 19897 19898 13149 13491 13002 30308
31923 24569 14689 10250 10311 10322 16090 16117 27878 26917 11186 11199
29949 16138 16143 17382 17389 17374 17426 17342 10891 12569 24230 24225
15977 25766 25160 25165 11861 21074 21056 13396 13400 13391 13402 12674
12657 12712 12696 31285 31286 24120 18320 18299 18308 18305 18330 18300
18363 12863 13893 13866 13876 13873 28227 31001 14215 14216 14207 17642
17640 14576 21037 18557 14529 14362 15715 15669 15670 17744 14594 14622
14586 17058 17063 17051 14677 14500 15208 15183 15162 15007 15024 12895
12871 12911 15131 15101 19030 15560 15547 30682 17536 17513 17505 22482
22445 15078 23787 23804 23778 23757 15290 14463 15783 15802 23030 17031
30984 15512 17761 17767 17837 17850 17848 27911 16239 16289 29828 28087
18777 18787 18791 21183 16780 16711 16703 16697 26002 16859 16858 27603
667 478 16824 29840 16902 15896 15911 20511 20508 29869 11930 11946
11997 11957 19202 19237 21892 17201 17188 17161 17446 17475 17085 17154
12740 18076 18065 18066 28595 28511 28611 18837 18840 18722 18679 18714
18743 18701 18687 16348 16360 16394 24735 24768 24712 18921 18960 33625
18945 18917 19094 19040 19109 12782 33615 33618 33623 33624 712 465
12810 12065 18215 18187 18166 18106 13983 13998 14044 18487 19392 19399
19419 19292 19308 19330 19864 19844 19874 23718 23717 23615 19478 19594
19695 19539 19574 31385 19763 9869 29924 25938 29918 29910 25010 24551
24504 24479 9709 9711 9681 20338 20993 20973 20424 20397 26642 25583
25543 25045 25060 25052 25046 25042 22717 21714 20811 20763 20596 20776
20599 32229 32230 23237 23197 23222 23219 30321 22363 22302 22378 10088
12282 24310 33403 33362 33411 33361 21602 21626 22394 31108 22229 22273
22276 22295 22840 22848 22864 21475 21440 21453 21315 21370 21364 21228
21259 21240 21252 24208 24170 23263 25915 24057 25924 23951 23999 23982
23972 23969 25730 25178 25733 25736 25734 26065 26087 26084 26079 24645
24643 24674 29993 23174 27476 27483 27492 25861 30071 25086 25827 25849
26600 26583 26592 24256 24281 28283 26951 26953 26946 26954 19780 25689
25675 27651 27681 27679 27702 27814 27720 27817 26571 25479 25424 25485
25449 11677 10738 10717 10699 10716 10667 10670 10785 27086 27072 27080
27042 27048 13770 25279 25273 25293 31043 31042 31050 26481 26523 26511
26403 26368 26200 26230 26353 26178 26188 27946 27932 27953 27285 27298
11367 11372 9507 9464 9454 9488 9510 12043 16496 30670 30647 30637
30645 12363 12411 12387 13318 13313 13322 13324 26140 26102 26098 16199
26123 26121 12531 24584 17918 13276 10383 10390 16067 16081 16062 16052
12604 12610 12024 12028 18642 18636 12634 12633 12242 12255 12258 12235
14917 14893 14926 10820 10817 10853 28194 28156 28173 28189 28037 27376
27379 27367 32499 32498 32497 28386 28119 28445 28469 28303 33180 28669
28668 9572 15275 17245 17680 29309 11661 28346 14403 31487 32812 30013
15272 30994 13492 31514 31515 28347 31513 27405 13647 13681 15274 24570
28670 32512 11816 28500 29711 29272 29698 9753 13265 13264 31578 32974
17603 10474 33620 10539 17584 33266 24116 11303 20364 16625 9971 24627
19515 13000 13001 14795 1210 871 907 906 905 1403 908 872
1825 1398 1402 1126 1545 1401 1153 1456 1251 1544 1376 1414
1527 1326 1382 1354 1494 1399 32976 25522 33052 10540 11571 11632
11634 477 11600 11588 11596 23063 22917 20960 2750 2321 3205 1897
4091 1848 3936 2108 3310 2266 2641 3109 3386 3273 2964 9770
2014 2319 2635 3299 2144 2961 2963 2013 2015 2898 10538 2993
1853 2061 2105 9769 3272 2485 2214 2718 2135 2962 2142 2617
2139 2254 2676 2490 2257 2965 9733 20083 18469 33515 33554 10949
14945 19522 32011 32013 32012 29071 21936 28993 11008 17663 17685 24855
20325 23293 15936 15935 16653 13487 14473 17870 19901 13486 9599 15423
33453 33451 33450 33452 33456 33454 33455 32395 23029 24572 23027 23028
19519 19520 32550 19516 32551 19518 32549 14435 19517 32975 11860 4790
5599 5598 4254 5710 4550 5166 6270 5985 6332 6216 5511 6418
21691 9260 8407 8946 9054 8794 9269 7826 6767 8162 7617 8699
8482 8879 8875 8872 8877 8873 8971 8874 8161 8605 20250 8876
8878 8917 8958 8970 6671 7328 9258 7480 8183 9259 7795 7671
15926 32496 11063 11062 11065 15273 33163 17783 23013 32475 14406 31124
31910 707 32116 32119 585 30443 33113 18995 18459 32546 19895 10874
15270 32419 30654 15061 25505 13004 14242 24815 33179 9800 33345 28864
15341 9856 23126 33630 713 28698 28760 28746 28764 16519 29808 16532
16579 16531 16569 12457 31192 31195 31200 17976 33060 31314 20966 24811
22095 21497 30954 30953 30914 29976 29980 11040 12095 31252 31271 12147
12136 12951 786 12994 10621 15302 10484 33622 708 571 26845 26850
33466 33468 31406 31398 28039 28042 28051 13156 13161 13195
760: 32673 33462 12213 27634 27613 31539 25803 10349 13101 31505 32924 32912
17681 18668 10626 10345 21672 33598 14953 23707 19274 14517 18628 17993
33505 19997 15594 17608 10241 10339 10340 23097 26609 10495 10275 10310
16104 16128 27874 11175 14422 25898 29947 24084 17393 17298 12568 24838
24227 24243 30005 21075 21080 26552 13409 19742 18331 18317 13851 11710
13860 26882 13228 14212 21039 18556 14374 14369 14366 27120 15698 15717
17740 14577 23896 17059 32887 14684 14504 15196 15035 12875 12909 32292
32298 32307 32601 32603 15109 15531 15526 17529 22492 22462 15084 23796
23817 32417 15794 17028 19928 15523 17795 17836 27894 28058 29821 16294
16291 28078 28226 18792 16789 16799 16790 16679 26001 16863 32374 27558
16823 16899 29891 11913 11903 19225 19266 21889 17182 17088 12732 18067
28603 18847 11158 18682 16342 24766 24738 18912 18930 19106 33618 12047
9873 18120 18182 14033 19336 19872 19849 19814 23621 19508 19676 19683
19596 20483 29913 20017 20007 21927 15746 24996 24361 9678 20983 20425
20563 26741 26739 26653 25607 25544 25038 22779 22716 22728 21720 21728
20787 32195 23179 12350 30342 30362 22355 22303 12315 33409 21613 21806
22227 22856 22857 21434 21480 21351 21397 21280 21222 24029 23260 29479
29489 25911 24070 28261 27190 23933 23958 25739 25170 26078 26057 24653
29984 25867 24672 27510 27490 25097 28289 30026 25127 26960 25681 27673
27796 27735 27747 25437 11680 11665 13046 11667 10744 24973 27102 13771
13759 25277 31047 26423 26436 26356 26267 26185 27945 27935 27973 27272
27278 11362 11368 9512 9538 11508 16454 30632 12384 13330 26095 16217
12518 10370 10411 30510 30515 30513 30505 30525 16070 12228 14942 10816
32463 24104 27984 27320 27377 18003 28408 28393 28385 28140 28127 28455
28428 28325 28323 17248 17574 17432 30767 32130 32128 10529 33495 33561
460 30395 13705 13651 17626 30766 20948 20531 12488 18461 14447 29704
10205 18015 15412 1565 940 1060 32038 17665 27443 18540 32399 14880
14883 11574 10628 3612 3736 2738 18393 22142 22158 22153 22157 29285
29096 14412 19769 10463 4913 4637 5635 5189 9206 9408 7484 6684
9239 6575 7989 9811 11814 22102 33033 32112 31913 33336 23708 21004
30746 14409 21553 28710 28708 23742 28632 16568 12864 12449 31196 31193
20526 32050 27143 31299 31329 20882 20877 20879 20866 21496 30933 30941
22109 11041 12118 12965 12988 10617 21749 21512 16599 12180 26863 31433
27532 13164 13193
761: 27649 27646 27638 30279 30267 30668 17711 14313 10321 10305 27865 29945
17303 12561 32127 11092 24234 21052 21062 21051 12671 12710 12705 22899
13852 20248 14208 20383 27121 15754 14604 33560 17057 14516 14474 14475
12934 12908 12885 32624 32632 15127 22489 22464 30983 17021 17847 17827
27905 27906 28100 21199 21181 16714 16688 16691 16861 16844 32361 25979
11959 21894 17197 17207 17115 16388 16389 18954 18962 18892 19104 19053
19064 18121 18205 14008 13980 14003 14001 19319 19315 29931 29933 20470
20460 20457 20018 20039 20040 9679 9690 20540 20557 26724 26654 26775
26727 25644 20779 32197 32224 30331 12305 12297 22847 21417 21424 21433
21409 21479 21350 21374 21303 21396 21279 21233 21215 23265 23267 23131
29482 24068 26052 26066 29982 24641 25875 24656 27495 27481 30090 30079
30150 27712 27675 25434 25321 25324 30159 13774 25281 31063 26494 26480
26232 26367 26376 26292 27937 27965 11793 9486 9466 16483 29745 16488
16222 26114 26113 16203 10386 10379 10381 30517 26016 12233 14915 11243
10819 10839 11248 28005 27997 27986 27375 28154 28121 20819 24107 14251
13516 15356 10814 33259 13690 13692 30735 16416 16419 29692 1120 952
1462 1882 2539 1881 2762 11561 28905 22145 29019 20297 20298 20296
29249 32405 24807 33217 23106 9942 5744 6146 4456 4739 15642 8665
7772 6984 6982 6983 7771 8290 8225 7773 8666 8558 9244 8806
7445 9372 7770 6796 8226 7444 8927 8289 6586 8693 7440 23081
31740 18857 9814 10630 17985 10551 11240 16766 30764 31458 18997 31737
31736 31876 33275 22133 21552 16514 10629 31227 31138 31317 20871 9963
21500 30915 30922 31603 22610 21758 21767 21757 15577 13190
762: 1022 1471 1061 1028 3558 2434 3668 29087 20377 6227 4182 5575
7908 7980 7981
763: 13440 27142 13441 27141 17604 31126 11210 29936 17341 25771 25770 18349
15172 12877 14151 17525 27584 25986 11978 18706 18924 12837 23753 19834
23628 23608 20064 30370 21631 22844 29484 27186 25110 25836 30034 25693
25450 11672 13767 26454 26515 26308 27952 27247 9461 30629 32477 28011
27312 28406 28490 14279 14428 31350 31351 31352 19994 9842 24823 16414
1192 949 3804 3367 2117 3093 2822 2587 2356 3803 1945 3143
3563 3440 3231 2159 3517 2006 31104 22056 15241 10587 12751 6311
5800 5745 6219 4538 6088 5294 6156 5451 7025 8351 14692 8283
7661 7361 6813 7369 6911 6812 7109 22921 24785 14468 28739 16516
31158 14429 30945 32656 31418 20120
764: 1780 3743 3742 21935 4893 5659 6368 4895 4894 6606 8099 8854
6590 27003 15470
765: 32534 17876 11118 1807 1259 1486 1457 2551 3400 2678 2679 2863
3176 3564 3789 2730 3090 3790 29148 5395 5394 6497 7583 8395
766: 23303 14764 11553 11554 22414 1045 24942 1954 3149 30024 19429 21905
20580 20582 33239 33240 33242 13085 11556 11557 20078 8467 6576 8495
7330 7146 11551 10482 10481 10480 18862 21913 21911 21914 24605
767: 9739 17635 30268 11117 9744 29187 23346 14743 17284 14390 10273 21778
11176 12723 24075 17292 30008 15244 13930 10937 17046 33021 15161 15005
12906 18536 23092 20939 17501 17514 17515 15076 23775 16987 17801 17793
16701 27534 11902 19197 18401 18990 9889 18141 13995 23541 19541 19554
19667 22187 24500 9700 9683 9685 9713 20395 20390 20420 20400 20399
25552 22787 32190 12479 21662 22079 22564 22566 21423 21408 21312 21302
21249 23259 23155 29481 23996 26054 27507 27498 25118 30147 25217 25229
30144 25214 25708 25462 11678 10701 25285 25270 25267 25264 26416 26312
26347 27983 29757 30618 16211 26009 12223 12845 10848 10843 11246 10831
10849 11252 10851 27993 27332 28430 13466 25351 13667 29710 16932 16930
15447 31988 938 1490 941 939 942 1152 11610 3829 3338 2147
2636 1983 2649 29073 27125 15350 4272 4712 4713 5057 4711 12317
26620 20709 10588 13665 10355 11884 11883 23830 30689 30688 28727 29811
16551 29817 12461 9784 30901 15480 29406 29405 11013 12092 22995 29407
28045
768: 20119 1345 1404 7250 7987 6703
769: 15737 17614 11764 1089 1615 3792 3791 28981 5627 4490 4195 6538
8304 9243 8059 6961 8342 7114 8060
770: 15472 15473 1625 1655 3626 2583 3318 5491
771: 23712 24438 1090 1158 1274 1764 20286 15504 5321 5960 7975 7633
7976 9270 7470 7974 7451 7973 6689 8755 8298 6850 7068 7275
6991
772: 33449 24954 28872 23556 23581 24330 24535 26667 26686 26791 25640 25657
22676 22631 30828 21212 19284 31921 28340 28503 13283 11257 11256 944
966 977 31340 31339 29635 24866 10954 31342 31341 18873 31344 31343
31345 15371 15242 10338 11549 32715 20959 18856 22831 18876 20708 13253
17897 22953 10970 10058 22071 22073 22072 22070 22066 22069 22068 22067
773: 1589
774: 25752 14973 28219 909 29246 24797 6318 20049 7876 6878 7359 15443
775: 23433 24327 22673 1459 10944 2914 3707 3706 4078 3319 22029 5736
5464 9096 6743 8124 6698 6699
776: 30699 30700 23635 24465 24150 24154 26715 22706 22759 24153 24157 22390
24151 24155 24152 24156 27468 23834 22897 23835 22936 1667 1668 22128
22127 22130 22129 23836 24149 3828 4045 2608 2328 1981 11289 31099
21984 21954 21953 29037 32028 28793 30198 18582 18583 18584 13635 10180
10179 5615 6083 5256 5570 6082 4890 23397 5364 8371 7901 7234
8812 9098 8459 7168 8610 7959 6775 7907 28235
777: 22614 28807 3067 1869 3264 3002 3265 3915 3914 3263 2644 33028
8912 23664
778: 22614 28807 1779 2395 33028 22082 6192 23664 33269 22937
779: 32557 15262 1114 1091 22889 29340 2367 2499 22890 22896 22888 33192
15074 6445 6491 7741 9001 8201 10054 11283 21582 22895 21581 22892
22893 11646 11648 11647
780: 17601 33118 31694 33559 27852 10525 28502 24912 19168 28534 28530 16365
18132 19302 23473 23546 23466 23634 23562 23577 24415 24507 24399 24450
24529 24414 25587 22704 11467 11327 15269 19457 19458 32939 14265 11649
20356 10147 22990 22991 33171 33168 33167 33166 33170 33169 11873 21739
27407 1721 30548 20195 15503 20189 31682 21675 19189 20006 24019 11069
30196 28943 29204 15886 15885 21928 31919 20921 20922 9569 23735 19706
15638 6102 5939 6774 8439 18870 7646 6643 7714 8438 18869 15875
9351 28810 28811 18503 30993 16677 15966 16907 15968 16906 15967 15969
16905 16909 16908 15960 15961 15963 15965 15962 15964 11882 31019 9826
17687 23294 24950 9448 17434 13261 33151 21699 21700 21698 12750 29677
19725 9967 9966 32582 32581 32593 19185 23089 15060 23093 10656 32466
11433 32938 20821 20820 10164 12848 31577 13962 30552 20710 20666 12752
25497 14830 25498 23652 20005 23342 15640 23651 20317 20830 20316 27406
24103 23012 14828 10526 33037 33124 33123 33121 15359 10813 33122 21176
21780 14876 32098 32063 32061 32062 32099 32097 32096 32054 32056 32055
32060 32101 32053 32058 10627 32059 32095 32052 32100 32051 24141 32057
30561 32505 21556 32048 23113 31860 9762 30493 30492 19914 20221 15305
20269 32415
781: 30842 25775 28224 23559 22278 11291 950 978 3567 2435 2337 3840
3841 3629 3566 3226 2796 22106 6199 5881 5178 6153 5486 6324
5236 5894 5694 9172 9171 7270 9170 8744 8956 8743 6713 7435
22391
782: 29578 31480 31483 31485 31484 31482 31481 31486 7173 24943
783: 10983 10996 10995 10984 10992 10994 10993 10997 10998 11006 11005 11004
11003 10987 10988 11002 11001 20062 14404 26989 10981 10980 10982 10991
10989 10990 23411 10986 1759 1407 1731 1524 1270 1694 1291 22939
17436 3126 2216 4006 2493 2702 3104 3557 2353 3934 3935 3163
3057 3933 2078 3538 10586 14239 31094 21980 18374 22019 21944 29068
24864 23292 12999 15331 20077 19460 13518 6084 5640 5258 4175 5159
5713 5157 4760 29415 5158 4617 8656 9290 9291 6953 8044 8315
8748 7389 8961 7735 7734 8275 7733 8043 10985 11000 10999 23110
784: 1426 1479 17615
785: 14103
786: 27437 23325 23313 12199 30262 9952 23316 586 720 824 756 33627
628 787 33621 792 613 33628 570 567 33619 814 815 794
9849 30995 32901 31135 17283 27845 14108 31006 30747 22061 9810 27871
26917 24092 16139 16156 17306 12641 18365 13911 14304 31863 14230 15676
14488 12907 18999 15551 14159 14149 17552 22481 23821 15288 23030 15510
17763 16240 18789 25993 32353 16843 16893 19204 21869 17156 17440 17145
18587 18613 12737 18077 28585 18839 18683 16310 19093 33615 12061 19369
19348 19881 19782 19618 29930 29915 20332 20991 21713 20780 32207 23190
30337 22315 22316 10093 12276 33372 21661 22267 22873 24194 24186 29487
23915 25169 25747 26088 25794 24668 25098 24287 24246 25787 28840 30151
25706 25483 10704 25306 27157 9449 11529 16486 12398 12498 24576 24590
17926 17922 13270 10364 16044 14939 10589 28200 28376 28440 33180 28669
28668 17680 29309 30013 30015 13709 31392 14451 11816 13265 13264 31578
17603 10474 33620 10561 10560 10559 14277 10539 16625 9971 24627 19515
15471 9994 29325 13000 13001 14795 32759 871 907 906 905 1403
908 872 1825 1398 1402 1126 1545 1401 1153 1456 1251 1544
1376 1414 1527 1326 1382 1354 1558 1494 1399 1370 1361 1341
1476 1587 1504 1817 1623 1264 1639 1417 1653 1543 1651 1590
862 1233 1377 1715 1400 33052 10540 23323 477 11624 23063 3499
4028 2641 3109 3386 3246 3273 2964 9770 2014 2319 2635 3299
2144 2961 2963 2013 2015 2898 10538 2993 1853 2061 2105 9769
3272 2485 2214 2718 2135 2962 2142 2617 2139 2676 2490 2257
2965 2543 2966 2289 2277 3328 2134 2143 1928 2724 1939 2130
2491 2967 1965 2140 2308 2313 2310 2128 2193 1998 1850 2480
1912 2131 2192 1851 2592 2241 2212 4052 1849 2316 2245 1996
4048 2784 2997 2968 2118 3086 2133 1839 2129 2297 2486 2176
4047 1894 2479 2172 2076 2727 1947 4046 2208 4050 3066 2141
1892 1843 1913 2242 2179 1937 2655 2251 2290 3500 2749 2132
3023 4032 2373 4069 1896 2492 2075 2279 2299 2171 2577 2320
2200 2155 2258 2180 4056 2174 1891 1966 4071 4057 1865 4037
4033 1842 2000 3073 1863 4049 2191 1899 2779 1943 2712 2234
2383 1841 9733 32132 23108 20083 18469 33178 14945 10076 13455 19522
32011 32013 32012 16981 20107 20325 23293 13487 23019 14473 19901 13486
15423 18523 10558 10557 20075 20076 33453 33451 33450 33452 33456 33454
33455 32395 23029 31809 23027 23028 20092 19519 19520 32550 19516 32551
19518 32549 14435 19517 23322 23317 10537 21855 10535 23677 23329 23676
23315 6270 6507 6332 6418 4301 4324 4726 4328 4329 5389 4354
5153 6314 6490 4783 6465 4472 4342 4859 10003 4218 4863 4216
4230 4448 4741 4325 4337 4219 4573 4411 4473 4330 4338 4213
4720 4513 4511 5388 5165 4341 4351 4214 4514 5244 4232 4949
5219 4451 4347 5218 5217 5216 4542 4231 4215 4343 4607 4340
5265 5148 4487 4217 4221 5146 5145 4452 5138 4353 5523 4483
4446 4574 5251 5252 5250 5247 5516 4786 4334 4333 4785 6464
6475 4705 5135 10005 4449 4947 6498 4925 4469 4413 5997 4946
4944 4941 4939 4929 4930 4928 4194 4919 4918 4327 4466 5144
32548 10534 23327 6476 4936 4934 4932 5478 5143 4572 4223 4943
4923 4484 23328 4727 4150 5573 4942 4922 4339 4886 4455 4453
5248 4323 6392 4512 5156 4445 5595 4945 4938 4415 4924 4352
4901 6093 23314 4608 33053 33203 4822 5524 5553 6480 5481 4395
10536 4427 5537 4447 10002 5482 5783 5162 5155 5139 4900 5246
4920 4716 5140 4857 4719 4948 4940 4921 5480 5137 4926 5142
6462 5463 5141 5249 4937 5163 5479 4718 5452 4220 4412 6043
4419 10004 4931 4486 4474 4530 4478 4454 4485 4565 4935 5173
4731 4717 5136 4933 4326 5154 5164 5888 4605 4927 4737 5171
6009 6451 5152 5477 4787 4784 4229 4450 4410 4376 5476 5170
6466 6218 4703 6463 5558 6235 5310 4704 4222 6003 6446 31946
23324 23318 23320 23319 23312 23311 23310 23309 8162 6947 8699 8482
8879 8875 8872 8877 8873 8971 8874 8161 8605 8876 8878 8917
8958 8970 7480 7795 7447 8208 7415 7210 7414 8349 8842 8445
8159 7417 8841 8511 8867 8157 8928 8916 8843 6730 6710 6594
6923 15926 9585 23895 21925 707 585 32546 23330 18427 23321 20085
33630 28779 17866 17865 17714 17713 18410 18409 18408 18412 18411 18407
31143 17936 30905 11049 12154 14305 22992 32134 22556 32131 23107 32321
32322 23109 32133 32137 32323 32136 32135 23326 26851
787: 17633 23325 23313 9952 23316 30105 13134 759 31135 17283 22061 26917
25151 21076 17066 15784 23030 478 33625 33623 25059 22249 25301 31070
33180 28669 28668 17680 29309 30013 30015 31392 14451 11816 13265 13264
31578 17603 10474 33620 10561 10560 10559 14277 10539 16625 9971 24627
19515 13000 13001 14795 871 907 906 905 1403 908 872 1825
1398 1402 1126 1545 1401 1153 1456 1251 1544 1376 1414 1527
1326 1382 1354 1494 1399 1370 1361 1341 1476 1587 1504 1817
1623 1264 1639 1417 1653 1543 1651 1590 862 1233 1377 1715
1400 33052 10540 23323 23063 2641 3109 3386 3273 2964 9770 2014
2319 2635 3299 2144 2961 2963 2013 2015 2898 10538 2993 1853
2061 2105 9769 3272 2485 2214 2718 2135 2962 2142 2617 2139
2676 2490 2257 2965 2543 2966 2289 2277 3328 2134 2143 1928
2724 1939 2130 2491 2967 1965 2140 2313 2308 2310 2128 2193
1998 1850 2480 1912 2131 2192 1851 2316 2592 2241 2212 4052
1849 2245 1996 4048 2997 2968 2118 3086 2133 1839 2129 2297
2486 2176 4047 1894 2479 2172 2076 4046 2727 1947 2208 4050
3066 2141 1892 1843 1913 2242 2179 1937 2655 2251 2290 2749
2132 3023 4032 2373 4069 1896 2492 2075 2279 2299 2171 2577
2320 2200 2155 2258 2180 4056 2174 1891 1966 4071 4057 1865
4037 4033 1842 2000 3073 1863 4049 2191 1899 2779 1943 2712
2234 2383 1841 9733 32132 23108 20083 18469 33178 14945 10076 13455
19522 32011 32013 32012 16981 20107 20325 23293 13487 23019 14473 19901
13486 15423 18523 10558 10557 20075 20076 33453 33451 33450 33452 33456
33454 33455 32395 23029 31809 23027 23028 19519 19520 32550 19516 32551
19518 32549 14435 19517 23322 23317 10537 21855 10535 23677 23329 23676
23315 6270 6332 6418 4324 4328 4354 5153 6314 6490 4301 5247
4783 6465 4472 4342 4859 10003 4218 4726 4863 4216 4230 4448
4741 4325 4337 4219 4573 4329 4411 4473 4330 4338 4213 4720
4513 4511 5389 5388 5165 4341 4351 4214 4514 5244 4232 5219
4451 4347 5218 5217 5216 4542 4231 4215 4343 4607 4340 5265
5148 4487 4217 4221 5146 5145 4452 5138 4353 5523 4483 4446
4574 5251 5252 5250 5516 4786 4334 4333 4785 6464 6475 4705
5135 10005 4449 4947 6498 4925 4469 4413 4949 5997 4946 4944
4941 4939 4929 4930 4928 4194 4919 4918 4466 5144 32548 10534
23327 6476 4936 4934 4932 5478 4327 5143 4223 4943 4923 4484
23328 4727 4572 4150 5573 4942 4922 4339 4886 4455 4453 5248
4323 6392 4512 4445 5595 4945 4938 4415 4924 4352 4901 6093
23314 33053 33203 4822 5553 6480 5481 4447 4395 10536 4427 5537
4608 10002 5524 5482 5783 5156 5162 5155 5139 4900 5246 4920
4716 5140 4857 4719 4948 4940 4921 5480 5137 4926 5142 6462
5463 5141 5249 4937 5163 5479 4718 5452 4220 4412 6043 4419
10004 4931 5171 4486 4474 4530 4478 4454 4485 4565 4935 5173
4731 4717 5136 4933 4326 5154 5164 5888 4605 4927 4737 6009
6451 5152 5477 4787 4784 4229 4450 4376 5476 5170 6466 4410
6218 4703 5558 6463 6235 5310 4704 4222 6003 6446 31946 23324
23318 23320 23319 23312 23311 23310 23309 8162 8699 8482 8879 8875
8872 8877 8873 8971 8874 8161 8605 8876 8878 8917 8958 8970
7480 7795 7447 8208 7210 8349 8842 7415 8445 7414 8159 7417
8841 8511 8867 8157 8928 8916 8843 6730 6710 6594 15926 9585
585 32546 23330 23321 713 16536 17866 17865 17714 17713 18410 18409
18408 18412 18411 18407 17936 21501 32134 22556 32131 23107 32321 32322
23109 32133 32137 32323 32136 32135 23326
788: 26640 26819 21171 14740 927 1073 3252 3785 3769 29079 28792 22049
30195 5621 4459 4319 4881 5584 7263 7163 8454 6572 8252 8103
31813 32253 16915 16914 15741 33480
789: 23076 13558 12208 30243 25063 13096 32931 31898 23659 13638 13641 13642
13643 10296 16103 16124 27858 27870 11204 24091 17336 12583 25767 21083
13405 12667 12708 11287 18341 11704 13846 14195 14350 17738 14613 14669
15165 15010 12921 32604 15092 15553 17557 15810 17015 15518 17815 28062
21185 16848 29856 11960 19243 17186 18054 28587 16362 24772 18909 19050
12068 18126 14016 18493 19395 19344 19877 19611 20473 25971 24476 9698
20982 20573 26779 25536 30108 22775 20794 32204 23223 12343 30355 22323
12273 33378 21633 22226 22879 21461 21368 21325 21234 21267 24054 24069
23910 25795 27494 30094 28267 25123 26957 25685 27695 27764 25438 10741
24982 27115 25292 31049 26478 26362 27938 27980 27241 9481 11466 16459
16484 30635 12404 13326 16221 16187 12495 10365 26008 12614 12230 14909
10836 27372 18011 28377 28138 28479 28314 13955 13669 30733 31280 20330
29721 1713 1726 1016 1432 1338 1388 1580 1171 1384 11568 3867
3866 29108 4144 6530 5662 4404 6634 7298 9223 32171 15974 22208
27428 20621 11434 14062 28745 16512 12458 31177 30887 12158 21750 21495
26836 31412 31893 28046 13639 13640 15389
790: 1284 1319 1383 1320 1406 1424 1485 29047 21951 30715 6350 5508
4497 4600 5375 6240 4163 5196 4594 5067 6077 5376 6299 5276
9148 8011 9151 9149 7507 9150 6925 7277 9218 7647 7648 9015
7209 18514
791: 1269 1212 1223 3777 3307 3778 2826 3306 2823 3199 3650 1845
4038 22037 4370 6453 5816 6452 4644 7045 9117 7473 9118 8024
7715 7043 7472 8692 9557 15732
792: 23325 23313 9952 23316 814 759 31135 17283 22061 26917 23030 33180
28669 28668 17680 29309 30013 30015 31392 14451 11816 13265 13264 31578
17603 10474 33620 10561 10560 10559 14277 10539 16625 9971 24627 19515
13000 13001 14795 871 907 906 905 1403 908 872 1825 1398
1402 1126 1545 1401 1153 1456 1251 1544 1376 1414 1527 1326
1382 1354 1494 1399 1370 1361 1341 1476 1587 1504 1817 1623
1264 1639 1417 1653 1543 1651 1590 862 1233 1377 1715 1400
33052 10540 23323 477 23063 2641 3109 3386 3273 2964 9770 2014
2319 2635 3299 2144 2961 2963 2013 2015 2898 10538 2993 1853
2061 2105 9769 3272 2485 2214 2718 2135 2962 2142 2617 2139
2676 2490 2257 2965 2543 2966 2289 2277 3328 2134 1928 2143
2724 1939 2130 2491 2967 1965 2140 2308 2313 2310 2128 2193
1998 1850 2480 2131 1912 2192 1851 2592 2241 2212 4052 1849
2316 2245 1996 4048 2997 2968 2118 3086 2133 1839 2129 2297
2486 2176 2479 4047 1894 2172 2076 2727 1947 4046 2208 4050
3066 2141 1892 1843 1913 2242 2179 1937 2655 2251 2290 2749
2132 3023 4032 2373 4069 1896 2492 2075 2279 2299 2171 2577
2320 2200 2155 2258 2180 4056 2174 1891 1966 4071 4057 1865
4037 4033 1842 2000 3073 1863 4049 2191 1899 2779 1943 2712
2234 2383 1841 2680 2164 1840 2170 4043 2119 9733 32132 23108
20083 18469 33178 14945 10076 13455 19522 32011 32013 32012 16981 20107
20325 23293 13487 23019 14473 19901 13486 15423 18523 10558 10557 20075
20076 33453 33451 33450 33452 33456 33454 33455 32395 23029 31809 23027
23028 19519 19520 32550 19516 32551 19518 32549 14435 19517 23322 23317
10537 21855 10535 23677 23329 23676 23315 6270 6332 6418 4324 4328
4354 5153 6314 6490 4301 4783 6465 4472 4342 4859 10003 4218
4726 4863 4216 4230 4448 4741 4325 4337 4219 4573 4329 4411
4473 4330 4338 4213 4720 4513 4511 5389 5388 5165 4341 4351
4214 4514 5244 4232 5219 4451 4347 5218 5217 5216 4542 4231
4215 4343 4607 4340 5265 5148 4487 4217 4221 5146 5145 4452
5138 4353 5523 4483 4446 4574 5251 5252 5250 5247 5516 4786
4334 4333 4785 6464 6475 4705 5135 10005 4449 4947 6498 4925
4469 4413 4949 5997 4946 4944 4941 4939 4929 4930 4928 4194
4919 4918 4466 5144 32548 10534 23327 6476 4936 4934 4932 5478
4327 5143 4223 4943 4923 4484 23328 4727 4572 4150 5573 4942
4922 4339 4886 4455 4453 5248 4323 6392 4512 4924 4445 5595
4945 4938 4415 4352 4901 6093 23314 33053 33203 4822 5553 6480
5481 4395 10536 4427 5537 4608 4447 10002 5524 5482 5783 5156
5162 5155 5139 4900 5246 4920 4716 5140 4857 4719 4948 4940
4921 5480 5137 4926 5142 6462 5463 5141 5249 4937 5163 5479
4718 5452 4220 4412 6043 4419 10004 4931 4486 4474 4530 4478
4454 4485 4565 4935 5173 4731 4717 5136 4933 4326 5154 5164
5888 4605 4927 4737 5171 6009 6451 5152 5477 4787 4784 4229
4450 4376 5476 5170 6466 6218 4703 5558 6463 6235 5310 4704
4222 4410 6003 6446 6400 6525 4468 4355 4477 31946 23324 23318
23320 23319 23312 23311 23310 23309 8162 8699 8482 8879 8875 8872
8877 8873 8971 8874 8161 8605 8876 8878 8917 8958 8970 7480
7795 8208 8349 8842 7417 8511 7447 8445 8159 8841 7210 7415
7414 8867 8157 8928 8916 8843 6730 6710 6594 15926 9585 585
32546 23330 23321 713 17866 17865 17714 17713 18410 18409 18408 18412
18411 18407 17936 32134 22556 32131 23107 32321 32322 23109 32137 32133
32323 32136 32135 23326 571 570
793: 20321 20360 32862 19178 23392 29004 22060 29239 28934 5045 9346 8107
7084 2074 2567 3096
794: 32564 13550 20245 33307 567 759 33341 18655 11205 17417 25756 21049
31288 18342 13010 10422 17048 15049 12897 15557 14155 22427 22473 15767
19927 28079 16793 32373 32349 27571 667 29859 29886 11898 17143 18620
18019 18086 28589 18669 16358 24713 18887 19124 14026 19819 19546 20345
22982 25894 24315 33436 21635 23011 23268 23142 24052 23990 30171 25830
24275 27690 27686 10713 25297 31056 26470 26512 26280 26298 26205 27236
9468 16455 12535 17911 17912 10973 17860 28038 28362 28480 10421 31502
17591 30666 18450 21141 33620 15384 14227 24140 24138 24615 32861 24635
1722 871 907 906 905 3596 3922 3298 4065 1999 3802 2112
2478 2458 3223 2907 3257 2855 21097 28638 32080 32030 28966 29316
28967 32081 13496 11639 5238 5239 4212 5954 5557 5953 4671 4167
5259 5281 6489 4211 5565 8406 8405 9287 8136 7241 7755 9166
7798 7244 8868 8679 8824 8504 7110 6838 7131 18502 707 19280
11091 9767 28686 29801 16557 12441 31171 31304 21519 24146 22203 11010
12088 786 24933 31413 28781 13137
795: 1813 858 1716 1482 1344 1734 885 877 3241 2618 2598 2597
2194 3026 1859 4291 4791 4434 4792 4793 4307 4364 4531 8302
7809 7407 8802 8840 8409 8125 9159 7408 7808 8803 6715 8939
7157 6729 6578 7760 6681 7906 6717 8568
796: 2470 2911 3434
797: 23595 24357 24395 26745 25551 22774 22931 32752 1004 1002 1003 1273
859 1232 1797 1679 1142 894 1724 1243 1418 920 12755 14420
2656 2077 13153 2639 3133 3134 2790 2725 2880 3621 2789 3055
2016 2791 3056 1944 3135 11160 13281 24109 20061 28345 29636 31619
14731 32944 5177 6298 4201 4738 5172 4526 5160 5418 6506 4755
4709 4714 4525 4742 4567 5417 4467 6070 4710 6205 5303 4756
8529 8443 8376 8378 8832 8375 8119 8377 8389 7609 8384 8392
8386 8385 8886 8379 8380 9032 8381 8383 7697 6690 7310 7396
8387 6563 7689 7930 7323 7941 7398 8001 8388 8079 7409 8382
7664 7637 7983 7622 6695 7676 6674 8747 8391 7385 8177 8689
8390 12490 20662 22159
798: 21160 23464 24408 24537 24340 26791 26667 25617 25636 25632 22643 22765
22766 32886 9795 23354 33524 28015 1127 31335 1979 1977 2465 3812
2463 2751 3710 32423 32422 19902 32983 10176 20086 15242 15371 23679
11429 23746 20853 5507 33095 33318 11328 20711 22972 9941 33059 10970
10964 14417 32181
799: 10487 23509 24363 26688 22762 24127 1512 916 3735 3102 3800 31653
5807 4781 6042 9980 20716 8548 7535
800: 954 970 913 1484 1974 3408 4335 5882 5356 5267 4399 6126
4118 8918 8819 8818 13625
801: 31499 13975 4044 3481 29024 22504 29120 29028 28950 29119 7999 6757
9796 15887 33562
802: 2819 2323 2096 3274 3456 29452 7682
803: 23407 17062 28074 23588 19495 24418 26769 25593 22668 23196 23210 24288
24266 25274 1627 1342 15976 3193 3797 2525 2161 2228 21959 5602
6501 5597 6500 8774 7650 7037 8645 8219 7038 8646 8535 22126
28242 20635 20622 28054 20111 17874 31698
804: 24131 3008 17721
805: 23603 1701 1514 1569 3577 4090 3645 3370 2175 3210 3188 3371
2974 3372 3646 6114 5811 8596 8255 8595 8566 24787
806: 23574 31925 17683 15727 20147 1329 993 15426 18850 32010 31004 5731
5951 6447 5413 6438 7307 6755 7303 8055 8361 7024 11226 3554
807: 33097 20849 27642 30282 30240 30224 13132 17606 26525 33232 33229 33222
9939 10524 11177 11201 14858 31781 31783 33333 13412 12700 18337 13878
11731 13896 32650 14210 23646 17743 17729 14617 14587 15152 14993 12916
12880 17826 32618 15096 23893 15555 22898 15276 23774 27914 28072 28055
16243 16297 28081 28102 28217 27150 29216 21896 31615 15634 26175 17151
28535 18718 16370 24709 24747 18971 19079 12786 12823 12082 9905 18137
18108 18209 18119 18157 18160 19374 19812 23468 19553 19534 19560 19549
19545 19646 19605 22188 9824 24558 20546 20542 20543 25555 25525 23736
22653 22743 22737 22647 23226 30353 33394 22078 23008 22224 22239 22238
32033 21407 21476 21301 21369 21229 23251 23138 23156 28262 28259 27194
23970 25180 26051 24657 25117 28291 28271 26963 25209 27685 27664 27677
27687 27688 27733 27743 27770 25331 25476 13022 10762 10726 10692 10757
27104 31045 26237 26284 26279 27977 27259 27274 29766 16472 29786 19904
10414 13333 26096 26144 12512 12529 16057 16068 12606 12026 18640 12626
12627 12625 12617 12243 14929 28199 27991 27373 27327 27342 28359 28431
28475 28444 28295 28312 18445 9763 11089 30537 30536 33218 33197 33225
33224 18434 29705 1260 1097 24955 11578 23112 3009 3906 2838 3702
3151 11562 31475 29262 15349 23740 13454 6306 4114 5427 6307 6539
4313 8373 8270 8374 7063 8642 20439 20690 13630 13668 24847 29502
29501 29492 29494 29500 29499 29496 29495 29498 29497 29493 16573 12442
33223 33230 33231 23763 31774 33508 31216 10127 33228 33226 33227 32314
30896 30935 29978 12144 33221 33219 33220 9846 31448 31410 31428 28783
808: 3618 2917 3062 2601 3633 3513 3635 3512 3958 3424 2929 3634
2998 6118 5100 4556 4864 6308 4917 6174 5644 4373 4510 5062
9081 9080 7986 7702 7984 8281 8223 7985 7130 8221 7268 6941
8134 7091 8542 7090 8520 8222 7769
809: 13619 30218 28222 18224 25563 25824 25305 26105 27378 1248 968 1434
1652 973 1470 1541 2818 3354 3422 29248 28939 29138 9366 16566
810: 10126 1015 1634 2483 3784 3533 2066 3730 2544 3734 2070 3466
3690 2788 2955 3302 3395 22493 29304 29303 29305 29299 29302 29300
29301 21905 20580 20582 15331 30470 30476 30475 30474 30473 30469 30471
30472 4990 8251 6784 6928 8953 6688 8551 8240 9104 9186 8980
7279 8492 8891 7181 6664 7175 6752 6888 18524 21914 21909
811: 28661 22081 32388 1700 4042 2309 2163 4106 4602 4876 5579 5769
5824 6940 8848 7991 24795 14778 20115 21697 15883
812: 10068 23518 24400 26713 25643 22795 10210 1519 1317 1279 2741 4094
2870 2982 3121 3122 3168 4080 4079 29026 5646 5854 4839 5282
4304 4829 16978 9173 8741 7070 8889 7088 9005 7634 22746 14258
10069 10149 32837 32838
813: 31895 31896 31897 12876 28112 15897 17447 18918 20560 12274 14918 16446
1792 2349 2942 2647 3020 2666 1875 3079 2568 20257 29182 20258
4187 4188 5991 6027 6454 4519 6832 8344 7362 8601 6726 18538
31894
814: 33064 31460 13083 14736 15482 13829 33154 33160 33152 33578 33579 33582
33583 33584 33585 33586 33587 33592 33590 33591 33593 33580 14177 10811
10807 13080 14083 10074 10055 10057 15321 15316 10056 14783 13580 13582
13587 13594 13588 13601 29353 15586 9927 9931 9935 9933 30720 15865
11167 9794 33576 33575 10063 11880 10062 11879 10060 10061 11877 11878
14337 25197 10237 10235 10234 10236 10231 10229 10230 31535 33542 33565
33566 11697 11698 33535 33540 33543 33544 9765 9995 33538 33541 33539
33548 33536 33546 33537 10438 33547 18251 18245 15486 19718 32105 10564
14067 13443 33343 13445 9932 9928 9930 9934 14172 14173 14174 14175
14178 14179 14180 14182 14183 14184 14186 14187 22043 9968 19140 22173
21737 30480 32669 19143 22175 19172 19173 22168 19171 22176 23078 22169
22177 10348 19282 22170 19175 22171 22812 31961 19184 22178 32784 19426
19430 31995 22179 22172 19473 22180 17566 20201 9566 20202 20026 20082
20192 31455 14068 14069 9828 9729 9728 9727 9726 15488 15490 31506
10567 9829 9831 9833 9835 9836 9830 9838 11396 11821 14302 11397
11398 11399 11400 11401 11403 11395 11404 11388 11390 11391 11392 11389
11823 11824 11825 11826 22563 11827 22087 11828 11829 13612 21776 10571
10568 10569 10570 19992 13603 20232 25369 20230 20233 11334 11335 11336
11337 11338 11339 11340 11341 11342 11344 11345 11346 11348 11349 11350
11351 11352 11353 11354 11355 11356 11357 11358 15327 11691 11699 11693
11694 11688 11654 10648 10649 13084 15314 20844 20845 15982 15985 19437
13212 13213 13214 11459 11460 11343 11347 14081 14082 19438 19445 13470
25149 13593 14075 14078 14079 11652 11653 11655 15997 32764 32774 32776
32780 32781 15322 15319 15320 16020 16016 16017 16018 16019 14784 15323
15329 15312 15325 15313 15311 24164 15500 15501 15502 31994 15568 14098
14102 13468 13471 13472 13473 14336 13502 13504 13507 13506 30739 13503
13505 13508 13501 13497 13511 13509 13498 13499 13510 13512 13513 13500
13827 13826 13828 13825 14113 23273 15310 15309 15324 15328 15318 15330
15326 15315 19470 22808 14779 14781 14782 14786 14780 14785 19439 19440
19447 32761 17259 20242 15799 15491 15489 20904 20901 20905 20234 19510
23274 20899 20903 20898 20897 23115 31507 10658 30738 26168 30740 20900
20603 20902 32325 20907 20908 20910 23833 23831 23832 22807 19711 32106
13266 13442 13444 19993 32775 32769 32792 32789 32799 32768 32791 32788
32796 20068 32770 32778 32782 32767 32787 20736 20740 20754 20755 20756
20739 30481 20757 20741 20742 20758 20743 20744 20745 20746 20747 20760
20749 20750 20751 20752 20759 20753 20738 20737 20748 30129 23276 23275
23272 13598 32785 32766 16736 22257 24163 24167 24168 31461 25510 25511
25254 25253 31034 31390 15866 26169 26170 26165 26166 26167 9865 23345
14101 30549 13081 28885 32786 28886 28887 28888 31550 28889 28890 23284
32793 32795 32800 32120 24817 24819 24820 24818 32948 25409 25410 25411
25412 25413 25414 25415 25416 25417 25418 25419 25420 25422 25423 23752
13477 31807 31806 15752 10801 31627 13262 10804 9953 27523 20434 32121
25509 29387 29648 29662 29642 29643 29644 29645 29646 29647 29649 29650
29651 29653 29654 29656 29657 29659 29660 29661 29663 29664 29665 29658
29666 29667 29668 29669 29670 29672 29673 25507 25508 29671 29388 10802
25421 22574 12753 15298 18433 10170 11163 11144 20240 25373 10165 10167
20239 10168 25370 10171 33041 10169 11219 23340 22811 20896 19174 20895
17941 32658 32779 32797 28630 27137 13602 20150 10602 22174 28891 32773
32803 17957 13590 19717 32657 25516 17942 19716 21508 33112 29652 29627
29625 29626 29624 32760 17489 19289 13583 13584 31459 20909 32805 13599
25405 20256 32783 29655 32823 20916 16731 16737 14076 20217 32552 31743
25515 16758 31744 9912 32488 32489 32802 32947 11696 23733 13267 25512
20906 32794 10533 31456 17279 33159 33158 26164 32771 32798 29371 10442
10450 31566 23730 31569 31564 31565 11440 31567 31568 31571 31572 13951
13633 13952 13953 13954 9565 32815 19449 13823 9921 9922 24160 31570
10803 32772 32790 32762 32763 32989 32765 21586 22626 10446 28662 9445
21505 32818 32819 32820 32821 32777 32822 32817 32659 17940 22905 10457
18017 13595 11734 17491 19422 22573 10443 10444 10449 10445 10447 10448
10451 10452 10454 10456 10455 10453 10458 10459 10863 15975 10810 16747
10493 14074 13832 13831 13834 13835 13837 13839 13836 9832 13838 13833
11051 18464 10809 11151 11152 10488 10489 10490 10491 9766 10492 15317
10808 11448 10583 14317 14181 16754 22932 10563 33596 16729 16728 16732
16730 16735 16733 16734 16738 16739 16740 16741 16742 16743 16744 16746
16745 16748 16750 16751 16752 16749 16753 16755 16756 16757 16759 16760
16761 16762 14419 16948 22903 23282 22902 23283 30479 14185 25377 23699
23308 14253 19281 22386 14249 13613 13788 17958 17959 17960 17961 10166
10532 9837 14418 25517 25513 23731 14321 19455 13600 13795 13794 14315
14319 14865 14867 14866 14861 23706 32806 23705 23702 23703 23700 23704
23701 23710 11695 11700 11692 11690 18447 32648 14848 14849 33153 33161
33156 33155 23300 10233 30721 14176 14077 14084 23114 15755 25372 18018
32801 32804 16526 24098 13589 32320 13581 23280 23281 13591 17267 17268
17269 17270 17271 9764 17272 13597 17487 17488 17490 11393 17492 23307
17493 17273 10562 9803 11820 22559 17686 17899 28646 25371 20238 25368
20231 20241 25378 20235 25374 20237 25375 25367 20236 25376 19526 19525
13790 13791 13596 25514 24097 24100 24099 25389 33545 25390 25392 25393
25394 25395 32950 25396 25397 25398 25391 20215 20216 20218 20219 13634
13592 13514 14080 28631 13824 28666 28667 24165 22131 28654 28648 11394
11402 28651 28655 28649 28650 28652 28656 28657 28658 28647 22904 31745
25400 30722 32949 32946 32951 32514 23301 9834 13585 10812 13586 24836
24837 15487 33577 31968 11822 31962 31963 32467 32468 32479 32480 32481
32482 32483 32484 32485 32486 25366 9929 10625 31512 31511 33567 27425
22809 31971 24162 24166 28663 28665 28664 15873 11689 13082 31964 24161
10240 10232 10238 10239 33157 31969 24169 31967 31966 32000 31970 31965
31972 23732 20048
815: 32258 32318 13571 17631 17932 15884 759 10600 31135 11112 18499 18500
31519 25401 17283 22611 19897 30308 31923 10250 10311 10322 27878 26917
16138 16143 10891 25766 25165 25160 11861 21074 21056 12674 12712 31285
12863 28227 31001 14215 14529 15670 15669 15715 17744 14500 15208 15131
15101 19030 15560 22482 22445 15078 23778 23804 23787 23757 15290 14463
15783 15802 23030 17031 30984 17761 17767 17850 17837 17848 27911 16239
29828 16289 28087 18787 18777 18791 21183 26002 16859 16858 667 16824
29840 15896 15911 19237 21892 17446 17475 17085 17154 12740 18076 18066
18065 28595 28611 28511 19094 19109 19040 12065 9904 19330 19292 19308
23718 23717 23615 19478 19695 31385 19574 19539 19594 9869 29924 25938
29918 29910 25010 24504 9709 20993 20973 20424 20397 25045 25060 25046
20811 23197 22363 22378 22302 10088 12282 22394 31108 22229 22273 24208
24170 25915 24057 24047 27416 23951 23982 25178 26065 24645 24643 24674
29993 26571 11677 10738 27048 27042 13770 31043 26481 26523 26368 26178
26188 27946 27932 27953 16496 30670 12531 24584 10383 10390 16067 12634
12633 12242 12258 10820 28194 28173 28156 28037 32499 32498 32497 28303
33180 28669 28668 17245 9572 17680 29309 31487 14874 32812 30013 27405
15274 28670 32512 11816 9753 13265 13264 31578 32974 17603 10474 33620
14277 10539 17584 1210 871 907 906 905 1403 908 872 1825
1398 1402 1126 1545 1401 1153 1456 1251 1544 1376 1414 1527
1326 1382 1354 1494 1399 1361 1370 32976 25522 33052 10540 11571
23063 9733 20083 18469 19522 32011 32013 32012 14473 17870 19901 13486
18523 20075 20076 33453 33451 33450 33452 33456 33454 33455 32395 23029
31809 24572 23027 23028 19519 19520 32550 19516 32551 19518 32549 14435
19517 32975 11860 5875 21691 7095 15926 32496 11063 11062 11065 11066
15273 33163 32475 14406 31124 30443 33113 18995 32546 15270 30654 24815
14242 15061 33179 9800 25505 15341 23126 16519 29808 16569 12457 17866
17865 17976 33060 17936 24811 22095 21497 30954 31602 11040 12095 31252
12147 31271 12136 10621 15302 10484 26845 33466 28042 28039 13161
816: 9778 19461 14276 3985 2460 28979 30205 30209 30206 22053 30210 29344
29343 14571 5205 5791 9373 7915 7086 9416 28803 31935 33191
817: 22815 10867 29459 20918 1688 10511 11103 29077 22042 19356 24940 4252
5545 4789 4121 4120 5631 4910 6541 6482 5855 5029 5750 31739
818: 30499 13547 13527 17636 17211 27636 25062 23661 27848 23098 10323 9984
11207 29938 17335 11852 24235 25163 30009 11709 13858 13941 13872 14218
21528 22393 15695 19188 19187 15150 15187 14991 15027 14976 12936 12888
32299 27147 14958 17499 14457 23772 30985 17802 17849 15644 27888 16232
16245 31667 16708 16775 20203 25997 32360 27583 27586 16828 29892 25980
11917 11963 19268 17181 17194 17089 12727 18671 20834 20835 18955 12794
12072 18128 18155 13986 14049 19832 23493 19613 25969 20038 24329 20432
20414 20409 20403 20530 26693 25540 25882 22672 22750 20796 20768 20814
20812 32201 23224 30324 22343 21627 22269 22272 22251 22837 21376 21295
21232 23134 23333 27432 27185 23912 26047 24649 25858 25113 24249 25783
27710 27759 25327 25469 11676 13047 13020 11669 11668 10709 27106 27054
31067 26418 26469 26212 26383 26191 27962 9456 9471 29761 30638 16207
26129 12496 12530 12497 10412 26007 12630 12232 12268 21918 28172 28025
27370 28484 28318 10576 32588 24568 20090 10788 14756 18013 13699 28509
29600 29687 17080 32269 963 19459 12481 30012 30011 31575 11580 28505
3830 3242 15216 28988 11868 23649 23648 23647 14467 11171 11301 5916
5180 4902 5179 4295 8726 6988 7231 6899 6627 8021 7116 22558
22926 29736 23650 9584 16934 19148 16633 24824 19081 33008 32518 33009
32519 18399 22615 17265 28732 29807 16510 16523 31159 30912 30883 30940
30846 31022 11038 12157 12140 12126 12115 19176 26871 31441 13204
819: 30246 25249 25240 27839 23239 14560 14558 14095 14724 16097 16123 25384
11192 16135 10885 12548 20091 14810 13427 12681 12718 18318 14331 18546
14523 16003 16004 16005 16006 16008 16007 14483 15169 15013 32628 19003
16261 25248 16667 16878 18624 21546 13636 21884 23695 17164 17456 18615
18724 16371 31958 12825 25241 25242 25250 19393 19318 19882 20344 20552
21721 12361 10085 12308 24198 30174 26085 23168 24255 28832 30146 24979
27064 25300 27161 27248 9523 11510 30640 12415 13346 26158 24581 17916
13279 14923 28204 18004 28456 25245 25246 25243 25244 735 1211 951
1698 10478 3911 3297 2401 2057 3674 3401 3311 2847 2410 3675
2380 2908 2849 3343 2403 4058 3455 3708 3050 2451 3583 2930
10873 14816 14817 19358 17285 32471 5705 4400 4315 6432 4844 6110
6331 5401 5501 5351 5402 6031 6032 7139 6981 8945 8705 9234
8094 6980 7207 9233 8096 7598 6780 7835 8241 6790 6629 7571
8668 21923 18426 33443 33438 33444 33442 33441 33439 33445 33440 33437
31488 29411 29410 12454 16923 17679 17678 16921 16919 16920 16922 31202
29166 29165 29164 29167 29163 21759 29155 26876
820: 33138 17600 23295 31235 12431 30249 30285 30286 30229 13110 19705 20735
21009 28880 28233 32870 14320 28877 28878 28879 31859 22093 18513 18512
15641 22819 33474 32168 9847 33174 13257 15229 10066 11738 17379 30002
13390 15135 14975 15563 33334 9761 25989 16819 16892 20510 11944 29595
18675 16344 24710 18936 18116 18219 19815 23444 23587 23545 19652 33347
21543 21544 30110 32203 32210 23214 12280 12316 22206 22285 22839 21451
21247 23157 27181 23943 30183 25102 30028 26945 25226 25474 27071 27041
13744 13745 26514 26286 27963 27307 11511 29755 16475 13312 16186 26155
26146 16185 24587 33061 27821 22818 31008 31010 28360 32879 21105 9956
18629 11641 16433 29280 22816 28334 15264 6404 5723 28333 5072 6503
5073 4461 6502 5020 4357 4401 7870 16952 21803 32264 16631 16630
15606 16303 13255 23898 16555 9841 30956 25383 714
821: 22934 22928 24101 23343 33186 10437 11146 23481 23510 24503 26637 26763
26716 25529 25533 25635 25585 22642 22791 16026 14572 22835 17718 17717
19911 11812 18237 29308 18235 1238 29307 18234 18236 1082 984 1429
1257 1533 1263 1055 1172 1165 1065 1493 1183 1452 1568 1654
18509 17705 11169 25386 3772 3393 3140 2512 32139 30702 21986 18394
5690 5319 4887 5301 8153 9022 8825 8662 7508 6592 7866 7867
24906 32750 32749 10080 19423 33034 23290 32111 15893 15266 33091 33533
27009 27008 10638 10636 10637 13231 20661 20688 20692 20624 14259 22948
10124 15499 15498 22167 31926 32895 23698 20933 10927 27461 31122 17045
30830 29264 29263
822: 23513 24401 26743 26740 25534 22690 1337 1473 2845 2461 2868 30748
20246 4318 4176 5968 6241 6256 6178 6378 9201 8195 10342 20639
18467 17618 24625
823: 11555 11554 11553 22414 29412 14396 1045 979 24942 3435 29320 20304
11556 32810 29207 18862 21913 14773
824: 13539 23325 23313 9952 23316 13121 759 31135 17283 22061 26917 25765
15782 23030 478 667 33625 30109 22279 31033 33180 28669 28668 17680
29309 30013 30015 31392 14451 11816 13265 13264 31578 17603 10474 33620
10561 10560 10559 14277 10539 16625 9971 24627 19515 13000 13001 14795
871 907 906 905 1403 908 872 1825 1398 1402 1126 1545
1401 1153 1456 1251 1544 1376 1414 1527 1326 1382 1354 1494
1399 1370 1361 1341 1476 1587 1504 1817 1623 1264 1639 1417
1653 1543 1651 1590 862 1233 1377 1715 1400 33052 10540 23323
477 23063 2641 3109 3386 3273 2964 9770 2014 2319 2635 3299
2144 2961 2963 2013 2015 2898 10538 2993 1853 2061 2105 9769
3272 2485 2214 2718 2135 2962 2142 2617 2139 2676 2490 2257
2965 2543 2966 2289 2277 3328 2134 2143 1928 2724 1939 2130
2491 2967 1965 2140 2308 2313 2310 2128 2193 1998 1850 2480
2131 1912 2192 1851 2592 2241 2212 4052 1849 2316 2245 1996
4048 2997 2968 2118 3086 2133 1839 2129 2297 2486 2176 4047
1894 2479 2172 2076 2727 1947 4046 2208 4050 3066 2141 1892
1843 1913 2242 2179 1937 2655 2251 2290 2749 2132 3023 4032
2373 4069 1896 2492 2075 2279 2299 2171 2577 2320 2200 2155
2258 2180 4056 2174 1891 1966 4071 4057 1865 4037 4033 1842
2000 3073 1863 4049 2191 1899 2779 1943 2712 2234 2383 1841
2680 2164 9733 32132 23108 20083 18469 33178 14945 10076 13455 19522
32011 32013 32012 16981 20107 20325 23293 13487 23019 14473 19901 13486
15423 18523 10558 10557 20075 20076 33453 33451 33450 33452 33456 33454
33455 32395 23029 31809 23027 23028 19519 19520 32550 19516 32551 19518
32549 14435 19517 23322 23317 10537 21855 10535 23677 23329 23676 23315
6270 6332 6418 4324 4328 4354 5153 6314 6490 4301 4783 6465
4472 4342 4859 10003 4218 4726 4863 4216 4230 4448 4741 4325
4337 4219 4573 4329 4411 4473 4330 4338 4213 4720 4513 4511
5389 5388 5165 4341 4351 4214 4514 5244 4232 5219 4451 4347
5218 5217 5216 4542 4231 4215 4343 4607 4340 5265 5148 4487
4217 4221 5146 5145 4452 5138 4353 5523 4483 4446 4574 5251
5252 5250 5247 5516 4786 4334 4333 4785 6464 6475 4705 5135
10005 4449 4947 6498 4925 4469 4413 4949 5997 4946 4944 4941
4939 4929 4930 4928 4194 4919 4918 4466 5144 32548 10534 23327
6476 4936 4934 4932 5478 4327 5143 4223 4943 4923 4484 23328
4727 4572 4150 5573 4942 4922 4339 4886 4455 4453 5248 4323
6392 4512 4445 5595 4945 4938 4415 4924 4352 4901 6093 23314
33053 33203 4822 5553 6480 5481 4395 10536 4427 5537 4608 4447
10002 5524 5482 5783 5156 5162 5155 5139 4900 5246 4920 4716
5140 4857 4719 4948 4940 4921 5480 5137 4926 5142 6462 5463
5141 5249 4937 5163 5479 4718 5452 4220 4412 6043 4419 10004
4931 4486 4474 4530 4478 4454 4485 4565 4935 5173 4731 4717
5136 4933 4326 5154 5171 5164 5888 4605 4927 4737 6009 6451
5152 5477 4787 4784 4229 4450 4376 5476 5170 6466 6218 4703
4410 5558 6463 6235 5310 4704 4222 6003 6446 31946 23324 23318
23320 23319 23312 23311 23310 23309 8162 8699 8482 8879 8875 8872
8877 8873 8971 8874 8161 8605 8876 8878 8917 8958 8970 7480
7795 7447 8208 7210 8349 8842 7415 8445 7414 8159 7417 8841
8511 8867 8157 8928 8916 8843 6730 6710 6594 15926 9585 32546
23330 23321 33630 713 16527 17866 17865 17714 17713 18410 18409 18408
18412 18411 18407 17936 32134 22556 32131 23107 32321 32322 23109 32133
32137 32323 32136 32135 23326
825: 9867 15214 32508 32541 31396 31395 33085 31631 13537 30220 14393 25814
13139 9806 14111 33353 10943 12589 25146 21079 12856 13949 12872 17253
18996 10855 22551 17500 15777 28210 27578 25985 29874 12002 18740 16356
20244 18122 18168 19316 23542 23536 23539 23478 23530 23435 23501 23477
23553 23415 19696 19626 19642 19542 20041 24423 24512 24424 24367 24428
24514 24331 24540 24515 24556 24457 24492 24446 24442 20387 26753 26781
26648 26789 26783 26759 26762 25566 25542 25040 22785 22786 22638 22735
22801 22680 22736 22666 22803 22687 22663 22800 22733 22744 22715 22664
20600 20797 32231 32216 22244 23242 24027 23133 23997 23921 23980 23938
23976 25748 24292 30154 27719 10687 13762 31074 26285 26259 9455 11537
30621 30674 12421 16179 12500 14922 28006 10941 10940 17689 853 2341
3487 2372 3917 3602 3268 3340 3662 21932 24676 29351 32409 14110
9949 4209 5848 9245 31496 20359 16951 11164 20700 14256 20695 20684
20719 33346 21091 16560 16518 12434 31215 20612 11014 12085 12093 12131
20122 15454
826: 24101 23343 33186 10437 23481 23510 24503 26637 26763 26716 25529 25533
25635 25585 22642 22791 16026 14572 22835 30667 32807 32808 19731 17718
17717 19911 11812 20328 21730 18237 18235 1238 29307 18234 1082 29308
18236 984 1429 1533 1263 1055 18509 17705 11169 3112 27404 32277
14800 14801 10008 31020 11121 4696 24906 32750 32749 10080 33034 23290
19423 32111 15893 15266 33091 33533 27009 27008 10638 10636 10637 13231
20661 20688 20624 14259 20692 22948 10124 15499 15498 22167 31926 32895
23698 20933 10927 31122 17045 30830 29264 29263 14745 17895 32510 23697
827: 33464 13219 27846 14423 16009 14332 10304 10246 19158 31629 19719 18304
11718 13892 12860 15168 15012 23770 30977 30975 17038 16988 17018 17825
17758 17819 27922 16280 16259 16258 16260 29824 28083 28106 28107 28111
28116 32355 20498 11985 19198 33211 18754 18752 18755 16383 18900 12836
12843 12777 12833 12783 12792 18111 18194 19293 19349 19334 19300 23505
23564 23491 19587 24452 24334 24462 24337 24359 24375 9684 9693 9710
9686 20426 26774 26704 26652 26643 26813 26786 26773 26639 26718 25557
22758 22681 22705 22767 20804 21652 22881 23984 23994 27504 27503 27680
25664 26485 26311 26378 11492 11491 30671 12397 16163 12267 12240 11251
10850 11245 10825 28192 28188 28012 21206 19766 13702 15924 19765 29735
29729 29680 19469 19767 19768 1246 1007 957 1775 1132 1575 1135
861 1561 921 1318 19710 29281 26915 19709 11607 3846 3314 20052
26884 29038 13476 20205 14633 4916 6021 7223 7724 6560 9820 32249
9819 9919 31703 29741 29170 18525 20664 21165 28812 33551 33278 28752
16506 29160 29159 29158 29162 29161 11042 12149 12984 12947 13215 28041
13187 13208
828: 17226 20243 13103 13092 23015 30695 32962 27016 29954 17395 17344 10911
21088 13864 11733 11724 13921 14205 14551 14382 17732 14621 23082 22576
15047 15102 19037 14128 15267 17824 27887 21186 16713 16690 27554 16898
29897 20519 11923 11894 18088 11131 16378 24755 24686 19113 12838 9881
13981 14002 14031 19887 19787 23431 23511 19483 19633 24502 24354 9701
26733 26730 26816 25630 22634 12475 12337 22364 33429 21596 22950 32032
21311 21355 21224 24066 25172 26064 26075 27487 27473 24290 24251 28286
25679 25210 27698 27760 25320 25316 10727 10786 10668 10758 24985 13773
26484 26483 26374 26249 26351 26240 27264 27302 11361 11370 9491 9539
9514 16463 30673 30617 30603 16193 10396 30504 16047 16073 16084 12637
12231 14913 10828 27387 28373 28387 28483 28317 10430 15372 9715 11618
11582 11633 22149 22147 5798 5280 5185 4523 4648 6159 9131 8618
7105 10915 9783 18858 13462 20686 20353 27469 28712 12451 31224 31153
31166 31296 31309 20858 25015 26835 31440 17929
829: 3442 4083 3365 5048 8485 7566 8135 8611 8670 7232
830: 21172 32522 28785 21173 2511 4084 29313 29368 29369 5363 5748 6247
831: 24553 22662 1148 967 3535 3536 3072 3520 3521 2593 32089 29191
4236 5923 5942 5821 8249 7729 7956 7420 20631 16624 14959
832: 18242 33208 18244 1195 1387 3283 18241 18243
833: 23508 24493 24364 26817 25581 15308 15306 15757 1464 10654 10655 32020
13804 14099 14106 16039 15954 29103 14250 32577 32579 32578 33030 32993
14433 21931 15481 8420 9181 8553 8287 8932 9132 7992 7488 9164
7016 23337 9940 32666
834: 22392 27515 1425 1436 2528 2186 3164 2783 3979 3980 2780 3088
3416 2431 3119 3415 2781 10950 29397 31772 6202 5229 5955 4125
4559 6414 4560 4558 6354 5956 5183 4627 4423 5184 4615 4471
8870 8120 7751 9121 8635 8636 8637 9092 15376
835: 26624 25526 22708 1510 2259 2626 3809 3745 2560 3092 3808 2629
28961 21960 5976 5967 5122 5998 5124 5125 5123 6461 4868 7863
7059 9108 8354 7880 7194 7882 7881 6739 20681 30212 15296 10919
30208
836: 23512 24443 26761 26766 25567 22771 22777 26910 1594 1083 3827 3238
3278 3355 1861 4025 28970 29044 29055 11140 4250 6092 5911 5466
5128 6468 6439 4508 7251 8035 8273 8644 7963 7823 8173 6939
6998 7089 8440 7492 6979 7020 9977 31536 9446 20657
837: 3462
838: 24435 26807 25569 22665 878 1230 1567 2386 4063 3236 1953 6005
4251 4369 6646 6869 9419 8994 6970 8853 8852 23080 10428 20650
15385 14776
839: 20536 17641 17639 23484 24336 24495 26738 26742 25554 25621 22674 19998
1525 3617 3598 3581 17684 9785 16937 23348 23347 5374 5889 6437
5527 6505 5785 7354 6647 7695 6786 7705 7787 33089 10329 16944
14868 11792 22094 20706 18533 14316 17263 33329 32278 15415 14833
840: 1276 1750 2527 2691 2396 3545 2243 3698 2526 3766 2685 1858
3464 3217 1987 2092 4422 4598 8054 7160 8736 7377 7919 8826
7667
841: 1265 1303 3402 2876 3505 2669 3611 3610 28920 23341 29085 6369
6296 4970 6155 5847 6053 7214 6724 6766 8833 9018 15416 15404
14163 29326
842: 23429 24531 25641 22671 19739 32832 1439 2759 3653 3137 2252 4061
2160 28791 28948 5768 5088 32712
843: 24760 12788 19866 23630 24333 26671 22782 25741 28378 15419 19164 21695
9562 15417 16642 22552 1770 1335 3198 3856 2352 2638 2956 2363
3751 2553 2552 3301 31927 20301 29029 11295 20449 22927 32869 20615
6654 6672 7315 7990
844: 25494 19361 22908 5607 4991 4204 5766 7666
845: 33086 13219 20847 18516 18767 10059 10799 27846 27836 18656 14423 10277
10285 12465 29593 17361 17373 17324 17398 17371 17333 10898 11233 13622
13890 12860 11718 13892 14206 14537 23645 15694 15700 15168 15166 14979
15012 19021 17558 17544 17556 17512 15289 23755 23791 15284 23812 30966
17797 16280 16781 16770 16660 32355 27596 27577 16836 20492 29871 20486
11966 11999 11951 19216 19242 17485 20263 33211 18725 16383 24780 24692
24781 18929 18925 12769 9907 18197 14043 14056 19889 19794 19890 23507
19576 19630 19679 25002 22977 20426 20804 12477 22356 12311 21600 21652
21634 22234 22881 24191 23164 28253 27201 27419 23935 23946 23984 23994
26050 25796 24667 25093 25838 30080 25074 30083 25073 30063 25114 30065
25091 25112 30142 25206 27680 27708 27785 27820 27819 27723 27742 27741
27804 27744 27727 27793 27772 25467 13034 26529 13737 13757 13743 13753
26415 26452 26460 26241 26370 26254 26215 26281 26348 27275 27292 27276
29775 16474 16461 29790 29788 29779 30671 12397 16177 31811 10382 12246
12221 12263 28170 28012 28403 11435 11545 33047 13259 9866 13674 24915
14392 15924 32274 16452 16415 16402 19469 17975 17582 4055 2873 14286
29349 22503 29101 22502 4878 6248 6186 5011 6477 5649 4552 5092
5449 5010 6424 6122 6054 4549 4496 5428 6420 4105 6315 4358
4753 4285 4524 4553 6516 5657 7237 7301 8904 7294 9067 9385
7586 7574 6803 9398 6930 7824 7615 8685 8448 8536 8777 8447
7425 7302 8191 10465 32249 20664 33551 13696 17439 31378 9964 31503
20262 28681 28735 28730 20367 28694 28729 16506 25359 33257 30934 30930
24911 15588 20917 11031 12125 12967 12981 10614 22996 13215 28041 13157
12464
846: 27631 27617 30263 30275 27471 9768 32586 15944 21775 32270 25905 25906
10908 24239 19444 19442 15268 19443 19446 19451 13408 13230 18336 13910
11728 14875 24987 21025 23642 31931 14490 15203 14985 12900 19436 19441
19448 31018 31017 31016 19010 15539 32123 14135 15803 30971 17809 27882
16717 32358 27570 19210 21856 17454 20604 16359 16353 18978 12808 12057
18195 13992 19314 19843 19880 19753 17982 20568 20818 23216 21620 21446
21336 23153 29486 23954 23963 26959 25701 25313 25447 11684 10733 30162
27105 27061 26438 26350 26186 27951 27220 9480 9517 12034 11540 29762
30592 13329 16204 16176 16173 26120 12521 26034 13485 28031 27987 28018
27328 27354 18010 20073 28356 28474 28310 11543 19454 19453 23353 9954
32122 9723 14335 14636 33040 28789 16404 32661 1010 31734 3348 3763
2700 2047 29372 9985 20726 23289 21530 19450 4978 4625 4171 5717
5235 5169 9204 7768 8465 9420 7557 18511 18465 14864 15872 19452
28733 31197 31176 31199 30907 31599 31023 26549 26548 26550 12129 12975
21745 16597 12178 22530
847: 12213 12204 27624 18668 10626 21672 33598 19274 14517 33505 15594 17608
10227 10241 10339 23097 30302 29568 11212 14422 24084 17354 17391 12546
24838 24227 24243 21075 21080 26552 13409 19744 18302 13900 13936 11710
13860 13901 13906 26882 13228 21039 18544 27120 17059 32887 27459 14504
15177 15179 15189 15018 15020 15029 12927 12932 32307 32298 32603 32601
15526 20106 15084 15285 32417 15770 15523 15514 16679 16799 16790 32374
27544 16823 16886 29867 20516 29884 20523 29887 11896 11996 11890 11887
12011 11900 21889 17182 17139 12725 16320 24765 24716 19054 12784 12821
12800 12053 18176 18131 18170 18135 18151 18134 14021 14045 14029 14033
18489 19871 19824 19838 23606 23414 19489 20475 20007 15746 24387 24373
24463 24539 20567 20563 26700 22716 21728 20767 12473 23203 12353 30319
22340 33383 21625 21611 21667 21806 22290 22227 22857 21430 21344 21266
23257 23260 25911 28261 27417 25170 25739 29984 24672 25119 25842 25862
25127 26960 27666 27691 27735 27781 25460 25425 25333 25436 11667 11670
13041 11665 10753 10766 10684 10750 10760 10763 10705 10686 10659 10751
10748 10789 24968 13759 25278 26435 26424 26510 26429 26518 26463 26461
26475 26214 26229 26270 26290 26338 26319 26250 26181 27271 27234 27228
9477 9465 9499 11493 12384 13330 16224 26092 10411 26024 26036 16043
16042 26041 26014 12635 12238 12244 28009 28016 27384 27358 27386 27362
28407 28422 28127 28493 28323 17248 17574 33495 33561 30395 13650 13688
30766 18504 24025 29725 29688 29703 10205 17586 11119 16638 10048 890
1506 32038 27443 18540 32399 14877 14883 11614 11637 3831 2926 18393
22153 22142 14412 19769 10463 5576 5577 9811 22924 31711 31710 31913
23177 23708 21004 30746 21553 30485 31875 28693 12864 31220 27143 21496
30941 22109 21747 21512 16599 12180 26863 31421 29374 8271 6929 7057
7134 9250 8046 8817 7010
848: 23424 23631 24458 24365 24542 26725 25648 25658 25561 22790 22732 11332
13079 15861 22122 32162 1121 1516 883 25335 26913 3890 26914 2270
2975 3467 2976 2574 3889 2114 2196 10573 5525 4862 33119 31695
18468 10548 14255 13463 30559 7333 22136 8673
849: 30228 30223 14367 14626 19301 29967 26073 25482 27924 9518 14925
850: 1133 1447 2484 3027 2104 1902 3091 3191 3156 2253 3451 3460
2436 2684 3469 2274 3465 1975 3423 2124 3656 2273 2169 2594
2756 2240 2437 3657 1883 2101 2595 2745 5859 5497 5187 5032
6150 6109 5454 6429 6374 5708 5517 5019 4577 8047 7281 7161
8192 7112 8830 7500 7686

Claims (12)

1. A plant cell nucleus with stably integrated, recombinant DNA wherein said recombinant DNA comprises a promoter that is functional in said plant cell nucleus and that is operably linked to a protein coding DNA encoding a glutamine synthetase protein having at least 90% identity to SEQ ID NO:478, and wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA and an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
2. The plant cell nucleus of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.
3. The plant cell nucleus of claim 2 wherein said herbicide is a glyphosate, dicamba, or glufosinate compound.
4. A transgenic plant cell or plant comprising a plurality of plant cells with the plant cell nucleus of claim 1.
5. The transgenic plant cell or plant of claim 4 which is homozygous for said recombinant DNA.
6. A transgenic seed comprising a plurality of plant cells with a plant cell nucleus of claim 1.
7. The transgenic seed of claim 6 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.
8. A transgenic pollen grain comprising a haploid derivative of the plant cell nucleus of claim 1.
9. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of recombinant DNA in a nucleus of claim 1, wherein said method for manufacturing said transgenic seed comprises:
(a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not contain the recombinant DNA, wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced high salinity tolerance, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil,
(b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants, and
(c) collecting seeds from selected plant selected from step b.
10. The method of claim 9 wherein said method for manufacturing said transgenic seed further comprises
(a) verifying that said recombinant DNA is stably integrated in said selected plants, and
(b) analyzing tissue of said selected plant to determine the expression or suppression of a protein having the function of a protein having SEQ ID NO:478.
11. The method of claim 9 wherein said seed is corn, soybean, cotton, alfalfa, canola wheat or rice seed.
12. A method of producing hybrid corn seed comprising:
(a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA in a nucleus of claim 1;
(b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA;
(c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide;
(d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants;
(e) repeating steps (c) and (d) at least once to produce an inbred corn line; and
(f) crossing said inbred corn line with a second corn line to produce hybrid seed.
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